Conjugates of cell binding molecules with cytotoxic agents

ABSTRACT

A conjugate of a potent cytotoxic agent with a cell-surface receptor binding molecule having a Formula (I), wherein T, L, m, n, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, and R13 are defined herein, can be used for targeted treatment of cancer, autoimmune disease, and infectious disease.

CROSS REFERENCE OF RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/253,881, filed on Apr. 16, 2014, entitled “CONJUGATES OF CELL BINDINGMOLECULES WITH CYTOTOXIC AGENTS,” which in turn is a continuation ofPCT/IB2012/053554, filed on Jul. 12, 2012. The entire content of each ofthe prior applications is hereby incorporated by reference.

1. FIELD OF THE INVENTION

This invention relates to a conjugate of a potent cytotoxic agent with acell-surface receptor binding molecule for targeted therapy. Theinvention also relates to use of the compositions comprising thecell-surface receptor binding molecule-antimitotic agent conjugates fortreating cancer, autoimmune disease, and infectious disease.

2. BACKGROUND OF THE INVENTION

There are many articles appeared on the attempted specific targeting ofpathogenic cells, in particular, cancer cells utilizing cytotoxic drugconjugated to cell-surface receptor binding agents, such as antibodies(Sela et al, in Immunoconjugates 189-216 (C. Vogel, ed. 1987); Ghose etal, in Targeted Drugs 1-22 (E. Goldberg, ed. 1983); Diener et al, inAntibody mediated delivery systems 1-23 (J. Rodwell, ed. 1988);Silverstein, Nat. Immunol. 2004, 5, 1211-7; Fanning et al, Clin.Immunollmmunopathol. 1996, 79, 1-14; Ricart A. D., et al., NatureClinical Practice Oncology 2007, 4, 245-255; Singh R. et Rickson H. K.,Therapeutic Antibodies: Methods and Protocols, 2009, 525, 445-467),folic acids (Sudimack, J. et al, Adv. Drug Delivery Rev. 2000, 41,147-162; Reddy, et al. Mol. Pharm. 2009, 6, 1518-25); PMSA (prostatespecific membrane antigen) binding ligands (Low, et al, WO 2009/026177A1); albumin with peptides (Temming, et al Bioconjugate Chem. 2006, 17,1385-1394); cobalamin and proteins (Gupta, et al, Crit. Rev. Therap.Drug Carrier Syst. 2008, 25, 347-79; Petrus, et al, Angew. Chem. Int.Ed. 2009, 48, 1022-8); carbohydrate (Darbre, et al, Curr. Top. Med.Chem. 2008, 8, 1286-93); bioactive polymers (Dhar, et al, Proc. NatlAcad. Sci. 2008, 105, 17356-61); dendrimers (Lee, et al, Nat.Biotechnol. 2005, 23, 1517-26; Almutairi, et al; Proc. Natl. Acad. Sci.2009, 106, 685-90); nanoparticles with binding ligands (Liong, et al,ACS Nano, 2008, 19, 1309-12; Medarova, et al, Nat. Med. 2007, 13, 372-7;Javier, et al, Bioconjugate Chem. 2008, 19, 1309-12); liposomes(Medinai, et al, Curr. Phar. Des. 2004, 10, 2981-9); viral capsides(Flenniken, et al, Viruses Nanotechnol. 2009, 327, 71-93), etc.

Different families of cytotoxic agents like calicheamicin derivative(Giles, et al Cancer 2003, 98, 2095-104; Hamann, et al, Bioconjug Chem2002, 13, 47-58), maytansin derivative (Widdison, et al, J Med Chem2006, 49, 4392-2408; Ikeda, et al, Clin Cancer Res 2009, 15, 4028-37;Xie, et al, Expert Opin Biol Ther 2006, 6, 281-91), auristatins(Sutherland, et al, J Biochem 2006, 281, 10540-7; Doronina, et al,Bioconjug Chem 2006, 17, 114-24), taxane derivatives (Miller, et al, JMed Chem 2004, 47, 4802-51; WO 06061258), leptomycine derivatives (WO07144709), CC-1065 and analogues (Suzawa, et al, J Control Release 002,79, 229-42; Suzawa, et al, Bioorg Med Chem, 8, 2175-84; WO 2007102069),doxorubicin (Trail, et al, Science 1993, 261, 212-5; Saleh et al, J ClinOncol 2000, 18, 2282-92), daunorubicin, vincristine, vinblastine,mitomycin C, or chlorambucil have been used for the conjugation with acell-surface receptor binding agent, in particular with antibodies (Wu,et al, Nat. Biotechnol. 2005, 23, 1137-1146. Ricart, et al, Nat. Clin.Pract. Oncol. 2007, 4, 245-255).

The use of a cell-surface receptor binding agent, particularly atargeting antibody having an affinity for the pathogenic cells makes itpossible to deliver the cytotoxic agent directly in the vicinity ordirectly in the pathogenic cell, thus increasing the efficiency of thecytotoxic agent while minimizing the side-effects commonly associatedwith the cytotoxic agents.

Several short peptidic compounds that found to have biological activityhave been isolated from natural sources. One of them, Tubulysins(structures shown below), which were original isolated by Hofle andReichenbach et al. (GBF Braunschweig) from a culture browth of the

Tubulysin R^(i) R^(ii) R^(iii) A CH₂OCOCH₂CH(CH₃)₂ OCOCH₃ OH BCH₂OCOCH₂CH₂CH₃ OCOCH₃ OH C CH₂OCOCH₂CH₃ OCOCH₃ OH D CH₂OCOCH₂CH(CH₃)₂OCOCH₃ H E CH₂OCOCH₂CH₂CH₃ OCOCH₃ H F CH₂OCOCH₂CH₃ OCOCH₃ H GCH₂OCOCH═CH₂ OCOCH₃ OH H CH₂OCOCH₃ OCOCH₃ H I CH₂OCOCH₃ OCOCH₃ OH U HOCOCH₃ H V H OH H Z H OH OH Pretubulysin CH₃ H H (The structures ofexisting tubulysin compounds)myxobacterial strains of Archangium gephyra (F. Sasse et al. J.Antibiot. 2000, 53, 879-885; WO9813375), are members of group ofantimitotic peptides that inhibit tubulin polymerization in dividingcells, and thus inducing apoptosis. With the exceptional potencyexceeding that of vinblastine, taxol and epothilones (Wipf, et al, Org.Lett. 2004, 6, 4057-60; Peltier, et al, J. Am. Chem. Soc. 2006, 128,16018-9; Wipf, et al, Org. Lett., 2007, 9, 1605-1607; Wang, et al, Chem.Biol. Drug Des. 2007, 70, 75-86; Pando, et al, Org. Lett. 2009, 11,5567-9), these antimitotic peptides are exciting leads for targetedtherapies. Structurally, the tetrapeptide tubulysins comprise ofN-methylpipecolinic acid (Mep) at the N-terminus, isoleucine (Ile) asthe second residue, the unique thiazole-containing tubuvaline (Tuv) asthe third residue, and two possible γ-amino acids at the C-terminus(tubutyrosine (Tut) or tubuphenylalanine (Tup)). Despite severaltubulysins have recently been synthesized, significant generaltoxicities (>15% animal body weight loss) of the existing tubulysins atdoses required for achieving a therapeutic effect compromise theirefficacy (US Patent appl. 2010/0048490). We have been interested in theart of a conjugate of a cell surface binding ligand, particularly usingan antibody to conjugate with tubulysin derivatives for havingsignificantly lower general toxicity, yet useful therapeutic efficiency.However, the tubulysins are hardly soluble in a buffer solution,resulting in significant amount of antibody aggregation when conjugatedwith the tubulysins. A simpler analog, such as using phenyl alanine(Phe) and tyrosine (Tyr) to replace Tup and Tut components respectivelyfor the antibody conjugation leads to the hydrolysis of Phe and Tyr inthe animal blood circulation and generates the much less potentMep-Ile-Tuv moiety (over 200 fold less potency than tubulysin A and D).Here this patent discloses conjugates of a cell surface binding ligandwith water soluble and stable, as well as in lower systematicaltoxicity, tubulysin derivatives and using these conjugates for treatingcancer and immune disorders.

3. SUMMARY OF THE INVENTION

In one illustrative embodiment of the invention provides a conjugate offormula (I):

and pharmaceutical acceptable salts and solvates thereof

Wherein T is a targeting or binding ligand; L is a releasablelinker; - - - - - is a linkage bond that L connects to a molecule insidethe round bracket independently; n is 1˜20 and m is 1˜10.

Inside the round bracket (parentheses) is a potent antimitoticagent/drug wherein R¹, R², R³, and R⁴ are independently C₁˜C₈ of alkyl,heteroalkyl; C₂˜C₈ of heterocyclic, carbocyclic, alkylcycloalkyl,heterocycloalkyl, C₃˜C₈ of aryl, Ar-alkyl, heteroalkylcycloalkyl,alkylcarbonyl; or two R's, such as R¹R², R²R³, R³R⁴, R⁵R⁶ and R¹²R¹³ canbe 3˜7 members of a carbocyclic, cycloalkyl, or heterocyclic,heterocycloalkyl ring system; Y is N or CH; In addition, R¹, R³, and R⁴can be H; and R² can be absent.

Wherein R⁵, R⁶, R⁸ and R¹⁰ are independently H, C₁˜C₄ of alkyl orheteroalkyl.

Wherein R⁷ is independently selected from H, R¹⁴, or —R¹⁴C(═O)X¹R¹⁵ or—R¹⁴X¹R¹⁵, wherein R¹⁴ and R¹⁵ are independently selected from C₁˜C₈ ofalkyl, or heteroalkyl; C₂˜C₈ of alkenyl, alkynyl; heterocyclic,carbocyclic, cycloalkyl; C₃˜C₈ of aryl, heterocycloalkyl, heteroaralkyl,heteroalkylcycloalkyl, alkylcarbonyl; X¹ is selected from O, S, S—S, NH,or NR¹⁴.

Wherein R⁹ is independently H, —O—, —OR¹⁴—, —OC(═O)R¹⁴—, —OC(═O)NHR¹⁴—,—OC(═O)R¹⁴SSR¹⁵—, OP(═O)(OR¹⁴)—, or OR¹⁴OP(═O)(OR¹⁵), wherein R¹⁴, R¹⁵are independently C₁˜C₈ of alkyl, heteroalkyl; C₂˜C₈ of alkenyl,alkynyl, heterocyclic, carbocyclic; C₃˜C₈ of aryl, cycloalkyl,alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl,alkylcarbonyl.

Wherein R¹¹ is independently H, R¹⁴, —R¹⁴C(═O)R¹⁶, —R¹⁴X²R¹⁶,—R¹⁴C(═O)X², wherein X² is —O—, —S—, —NH—, —N(R¹⁴)—, —O—R¹⁴—, —S—R¹⁴—,—S(═O)—R¹⁴—, or —NHR¹⁴—; R¹⁴ is C₁˜C₈ of alkyl, heteroalkyl; C₂˜C₈ ofalkenyl, alkynyl, heterocyclic, carbocyclic; C₃˜C₈ of aryl, cycloalkyl,alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl,alkylcarbonyl; R¹⁶ is H, OH, R¹⁴ or one to four amino acid units;

Wherein R¹² is independently R¹⁴, —O—, —S—, —N—, ═N—, ═NNH—, —NH(R¹⁴)—,—OR¹⁴—, —C(O)O—C(O)OR¹⁶—, C(O)NH—, C(O)NHR¹⁴, —SR¹⁴—, —S(═O)R¹⁴—,—P(═O)(OR¹⁶)—, —OP(═O)(OR¹⁶)—, —CH₂OP(═O)(OR¹⁶)—, —SO₂R¹⁶—. R¹⁴ isindependently C₁˜C₈ of alkyl, heteroalkyl; C₂˜C₈ of alkenyl, alkynyl,hetero-cyclic, carbocyclic; C₃˜C₈ of aryl, cycloalkyl, alkylcycloalkyl,heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkyl, alkylcarbonyl.R¹⁶ is H, OH, R¹⁴ or one to four amino acid units;

Wherein R¹³ is C₁˜C₁₀ of alkyl, heteroalkyl, alkyl acid, alkyl amide,alkyl amine, or Ar; Ar refers to a aromatic or hetero aromatic group,composed of one or several rings, comprising four to ten carbon,preferentially four to six carbon atoms. The term of hetero aromaticgroup refers one or several carbon on aromatic group, preferentiallyone, two or three carbon atoms are replaced by O, N, Si, Se, P or S,preferentially O, S, N. The term aryl or Ar also refers to a aromaticgroup, wherein one or several H atoms are replaced independently by R¹⁷,F, Cl, Br, I, OR¹⁶, SR¹⁶, NR¹⁶R¹⁷, N═NR¹⁶, N═R¹⁶, NR¹⁶R¹⁷, NO₂,SOR¹⁶R¹⁷, SO₂R¹⁶, SO₃R¹⁶, OSO₃R¹⁶, PR¹⁶R¹⁷, POR¹⁶R¹⁷, PO₂R¹⁶R¹⁷,OP(O)(OR¹⁷)₂, OCH₂OP(O)(OR¹⁷)₂, PO(OR¹⁶)(OR¹⁷), OC(O)OP(O)(OR¹⁷)₂,OP(O)(OR¹⁷)OP(O)(OR¹⁷)₂, OC(O)R¹⁷ or OC(O)NHR¹⁷, wherein R¹⁶, R¹⁷ areindependently H, C₁˜C₈ of alkyl, heteroalkyl; C₂˜C₈ of alkenyl, alkynyl,heterocyclic, carbocyclic; C₃˜C₈ of aryl, cycloalkyl, alkylcycloalkyl,heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl orC₄˜C₁₂ glycosides, or pharmaceutical salts.

In addition, R¹² can be H when R¹⁰ is not H, or when R¹³ is:

wherein Z¹ is H, CH₂OP(O)(OR¹⁸)₂, PO(OR¹⁸)₂, C(O)OP(O)(OR¹⁸)₂, C(O)R¹⁸,C(O)NHR¹⁸, SO₂(OR¹⁸), C₄˜C₁₂ glycosides or C₁˜C₈ of alkyl, carboxyalkyl,heterocyclic; R¹⁸ is H, C₁˜C₈ of alkyl, carboxyalkyl, heteroalkyl; C₂˜C₈of alkenyl, alkynyl, heterocyclic; C₃˜C₈ of aryl, alkylcarbonyl; R¹⁹ isH, OH, NH₂, OSO₂(OR¹⁸), XCH₂OP(O)(OR¹⁸)₂, XPO(OR¹⁸)₂, XC(O)R¹⁸,XC(O)NHR¹⁸, C₁˜C₈ of alkyl, carboxyalkyl, carboxylic acid derivative;C₂˜C₈ of alkenyl, alkynyl, heterocyclic; C₃˜C₈ of aryl, alkylcarbonyl;or pharmaceutical salts; X is O, S, NH; Y¹ and Y² are N or CHrespectively.Or R¹² can be H when R¹¹ is:

X² is O, S, N—R⁸; R⁸ is H, C₁˜C₆ of alkyl or heteroalkyl.

In another embodiment, the linker L of the potent antimitoticagent-binding molecule conjugates has the formula: -Ww-(Aa)r-Vv-;wherein: -W- is a Stretcher unit; w is 0 or 1; each -Aa- isindependently an Amino Acid unit; r is independently an integer rangingfrom 0 to 12; -V- is a Spacer unit; and v is 0, 1 or 2. The Stretcherunit W may independently contain a self-immolative spacer, peptidylunits, a hydrazone bond, disulfide or thioether bonds.

In another embodiment, the cell-surface binding molecule T may be of anykind presently known, or which become known cell binding ligands, suchas peptides and non-peptides. Generally the binding molecule T is anantibody; a single chain antibody; an antibody fragment that binds tothe target cell; a monoclonal antibody; a single chain monoclonalantibody; or a monoclonal antibody fragment that binds the target cell;a chimeric antibody; a chimeric antibody fragment that binds to thetarget cell; a domain antibody; a domain antibody fragment that binds tothe target cell; adnectins that mimic antibodies; DARPins; a lymphokine;a hormone; a vitamin; a growth factor; a colony stimulating factor; or anutrient-transport molecule (a transferrin); a binding peptide, orprotein, or antibody, or small molecule attached on albumin, polymers,dendrimers, liposomes, nanoparticles, vesicles, (viral) capsids.Preferably the binding molecule T is a monoclonal antibody.

In yet another aspect, a compound of formula I˜VII or a pharmaceuticallyacceptable salt or solvate thereof is used for treating cancer, anautoimmune disease or an infectious disease in a human or an animal.

4. A BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the synthesis of branched linkers for conjugation of drugsto cell binding agents.

FIG. 2 shows the synthesis of maleimido linkers and their use for thedrug-binding molecule conjugation.

FIG. 3 shows the synthesis of bromomaleimido and dibromomaleimidolinkers and their uses for the drug-binding molecule conjugation.

FIG. 4 shows the synthesis of amino acid (Val-Cit) linkers for theconjugation antimitotic agents with a cell surface binding ligand.

FIG. 5 shows the synthesis of Tuv component of the antimitotic agents.

Conditions: a. CuSO₄, H₂SO₄, Acetone, 95%; b: DIBAL-H, THF/Tol, −78° C.,95%; c: NH₂OH, NaHCO₃, CH₃OH/H₂O; d: isobutyraldehyde, MgSO₄, CH₂Cl₂,85% (2 steps); e: (2R)-N-(acryloyl)bornane-10,2-sultams (74), 40° C., 48h, CH₂Cl₂ 83%; f: LiOH, THF/H₂O, 86%; g: HClO₄, CH₃CN/H₂O, 98%; h:BOC₂O, Na₂CO₃, THF/H₂O, 95%; i: L-(S)-Tr-cysteine methyl ester, EDC,CH₂Cl₂, 85%; j: Ph₃P═O, Tf₂O, CH₂Cl₂; k: MnO₂, CH₂Cl₂, 76% (2 steps); l:Mo(CO)₆, CH₃CN/H₂O, 87%; m: Ac₂O, Pyr., 95%; n: NaH, THF, CH₃I, 85%; o):HOSnMe₃, ClCH₂CH₂Cl, 80° C.; 95%.

FIG. 6 shows the synthesis of Tuv components of the antimitotic agents.

Conditions: o): Fmoc-Cl, NaHCO₃, THF/H₂O, 95%; p): L-(S)-Tr-cysteinemethyl ester, EDC, CH₂Cl₂, 87%; q): Ph₃P═O, Tf₂O, CH₂Cl₂; r): MnO₂,CH₂Cl₂, 76% (2 steps); s): Mo(CO)₆, CH₃CN/H₂O, 87%; t): TES-Cl, Pyr.,95%; u): NaH, THF, CH₃I, 90%; v): NaH, THF, BrCH₂COOtBu, 0° C., 87%; w):HOSnMe₃, ClCH₂CH₂Cl, 80° C., ˜90%; x): Bu₄NF, THF; y): Ac₂O, Pyr. 81%.

FIG. 7 shows the synthesis of Boc-Tuv moieties and a conjugatableantimitotic agent.

Conditions: a): NaH, THF, N-(4-Bromobutyl)phthalimide, NaI, ˜83%; b);NaH, DMF, CH₃I, 90%; c): HOSnMe₃, ClCH₂CH₂Cl, 80° C., ˜85%; d): Ac₂O,Pyr.; e): (R)-(+)-b-Methylphenethylamine, EDC, DMA, 85%; f): 4M HCl,dioxane; g); Boc-Ile-OH, PyBroP, DMAP, DMA, 78%; h): D-Mep,PyBroP/CH₂Cl₂, 81%; i): NH₂NH₂, DMA; j); 58 (n=3), EDC, DMA, k): Ac₂O,Pyr. 56%.

FIG. 8 shows the synthesis of Tuv, Ile-Tuv and Mep-Ile-Tuv moieties ofthe antimitotic agents.

FIG. 9 shows the synthesis of Ile-Tuv, Mep-Leu-Tuv, Val-Ile-Tuv andVal-Ile-Tuv (O-alkyl) moieties of the antimitotic agents.

FIG. 10 shows the synthesis of a conjugatable antimitotic agent and itsconjugation to an antibody.

FIG. 11 shows the synthesis of amino acid (Phe-(D)Lys) linkers forconjugation of the antimitotic agents.

FIG. 12 shows the synthesis of antibody-antimitotic agent conjugates.

FIG. 13 shows the synthesis of antibody-antimitotic agent conjugates.

FIG. 14 shows the synthesis of the binding molecule-antimitotic agentconjugates.

FIG. 15 shows the synthesis of a binding molecule-antimitotic agentconjugate.

FIG. 16 shows the synthesis of an antibody-antimitotic agent conjugate.

FIG. 17 shows the synthesis of an antibody-antimitotic agent conjugate.

FIG. 18 shows the synthesis of hydrophilic Tut analogs for the synthesisof the hydrophilic (phosphate prodrug of) antimitotic agents.

FIG. 19 shows the synthesis of the conjugates of the antimitotic agentswith an antibody.

FIG. 20 shows the Boc solid-phase synthesis of the conjugatableantimitotic agents.

Conditions: a): Piperazine (5˜20 eq), CH₂Cl₂, 4 h; b): Boc-Aa₂-OH (2˜5eq), PyBroP (2˜5 eq), DIPEA (3˜10 eq), DMF, 4 h; c): 4M HCl/Dioxane, 0.5h; then washed with DIPEA (2˜3 eq), DMF; d): Boc-Aal-OH (2˜5 eq), TBTU(2˜5 eq), DIPEA (3˜10 eq), DMF, 4 h; e): BocNMe-Phe-OH, or Boc-Trp-OH,or BocNMe-Tyr(PO(OBz)OH)—OH (2˜5 eq), or BocNMe-(Pyr)Ala-OH, orBoc(Thienyl)Ser-OH, or Boc-(Thiazolyl)-Ala-OH, PyBroP (2˜5 eq), DIPEA(3˜10 eq), DMF, 4 h; f): Boc-N(Me)-Tuv-OH (1.5˜3 eq), PyBroP (2˜5 eq),DIPEA (3˜10 eq), DMF, 2 h; g): Boc-Ile-OH (2˜5 eq), PyBroP (2˜5 eq),DIPEA (3˜10 eq), DMF, 3 h; h): NMe₂-Ile-OH, TBTU (2˜5 eq), DIPEA (3˜10eq), DMF, 2 h; i): TFA, anisole; j): 4-maleimido butyric acid NHS ester(1.5˜2 eq), DIPEA (3˜10 eq), DMF, 2 h; k): 4-(methyldisulfanyl)butanoicacid NHS ester (1.5˜2 eq), or 4,4-dimethyl 4-(methyldisulfanyl)-butanoicacid NHS ester (1.5˜2 eq), DIPEA (3˜10 eq), DMF, 2 h; l): TCEP (3˜10eq), Dioxane/buffer pH 7.0, then solid supported guanidine.

FIG. 21 shows the Fmoc solid-phase synthesis of the conjugatableantimitotic agents. Conditions: a): (MeNCH₂)₂ (5˜20 eq), DCM, 4 h; b):Fmoc-Aa2-OH (2˜5 eq), PyBroP (2˜5 eq), DIPEA (3˜10 eq), DMF, 4 h; c):20% piperidine, DMF, 2 h; d): Fmoc-Aal-OH (2˜5 eq), TBTU (2˜5 eq), DIPEA(3˜10 eq), DMF, 4 h; e): (2˜5 eq) FmocNMe-Tyr(SO₃H)—OH, orFmoc-TrpOH—OH, or FmocNMe-Tyr(PO(OBz)-OH)—OH orFmocNMe2-Tyr(Glucose)-OH, or Boc-(quinolyl)Ala-OH, orFmoc-(thieny)Ser-OH, PyBroP (2˜5 eq), DIPEA (3˜10 eq), DMF, 4 h; f):Fmoc-N(Me)-Tuv-OH (1.5˜3 eq), PyBroP (2˜5 eq), DIPEA (3˜10 eq), DMF, 4h; g): Fmoc-Ile-OH (2˜5 eq), PyBroP (2˜5 eq), DIPEA (3˜10 eq), DMF, 4 h;h): Mep-OH (2˜4 eq), or NMe₂Leu-OH, TBTU (2˜5 eq), DIPEA (3˜10 eq), DMF,2 h; i): TFA, DCM, anisole; j): 4-maleimidobutyric acid NHS ester (1.5˜2eq), DIPEA (3˜10 eq), DMF, 4 h; k): 20% TFA, DCM; l):4-(methyldisulfanyl)butanoic acid NHS ester (1.5˜2 eq), or 4,4-dimethyl4-(methyldisulfanyl)-butanoic acid NHS ester (1.5˜2 eq), DIPEA (3˜10eq), DMF, 4 h; m): TCEP (8 eq), Dioxane, buffer pH 7.0, then solidsupported guanidine.

FIG. 22 shows the synthesis of the hydrophilic antimitotic drugs andtheir conjugates with an antibody.

FIG. 23 shows the synthesis of Tuv derivatives and the solid phasesynthesis of components of Mep-Ile-Tuv and NMe₂-Val-Ile-Tuv.

Conditions: a): compound 72, DIPEA, CsI, DMF, 2 h; b): 20% TFA/DCM, 0.5h, then washed with DIPEA, MeOH, DCM; c): Boc-Ile-OH (3˜5 eq), PyBroP(3˜5 eq), DIPEA (3˜10 eq), DMF, 6 h; d): Mep-OH (2˜4 eq), or NMe₂Leu-OH,TBTU (2˜5 eq), DIPEA (3˜10 eq), DMF, 6 h; e): 95% TFA/anisole/DCM.

FIG. 24 shows the synthesis of the antimitotic agents and theirconjugation with an antibody.

Conditions: a): (COCl)₂ 6 eq, DMF (cat), DCM, 1 h; b):D-(+)-Boc-norephedrine 4 eq, DIPEA, DCM, 4 h; c): 20% TFA/DCM, 0.5 h,then washed with DIPEA, MeOH, DCM; d): compound 274 (1.2 eq), TBTU (5eq), DMF, 6 h; e): Boc-Ile-OH (3˜5 eq), PyBroP (3˜5 eq), DIPEA (3˜10eq), DMF, 4 h; f): Mep-OH (2˜4 eq), or NMe₂-Leu-OH, TBTU (2˜5 eq), DIPEA(3˜10 eq), DMF, 2 h; g): HOSnM₃, ClCH₂CH₂Cl, 80° C., 8 h.

FIG. 25 shows the solid phase synthesis of antimitotic agents and theirconjugates with an antibody.

Conditions: a): 20% TFA/DCM, 0.5 h, then washed with DIPEA, MeOH, DCM;b): Boc-tiazolyl-Ala-OH (2 eq), PyBroP (4 eq), DIPEA (4 eq), DMF, 6 h;c): compound 276, TBTU (4 eq), DIPEA (4 eq), DMF, 6 h; d): Boc-Ile-OH (4eq), PyBroP (4 eq), DIPEA (4 eq), DMF, 6 h; e): Mep-OH (2˜4 eq), orNMe2-Leu-OH, TBTU (2˜5 eq), DIPEA (3˜10 eq), DMF, 2 h; f):TFA/DCM/anisole/p-thiolcresole (95:4:0.5:0.5).

FIGS. 26A-26B shows the solid-phase synthesis of antimitotic drugs andtheir conjugates with an antibody.

Conditions: a: DMF/Piperidine (4:1); b: 331/DMF/PyBroP (2˜5 eq); c:Fmoc-Tuv-OH (1.2 eq), TBTU (5 eq), DMF; d: Fmoc-Ile-OH (4 eq), PyBroP (4eq), DIPEA (4 eq), DMF; e: N,N (methyl, Maleimido-pentanoic)-Val-OH (2eq), TBTU, DMF; f: N,N-(methyl, 2′-pyridinyl-disulfanylbutyric)-Val-OH,TBTU (4 eq), DMF; g: 5% TFA/DCM/l % TIS; i: DTT/pH 7.0 PBS buffer/DMF,then HPLC; j: N, N (methyl, Maleimido-pentanoic)-Mep-OH (2 eq), TBTU,DMF; k: N,N-(methyl, 2′-pyridinyl-disulfanylbutyric)-Mep-OH, TBTU (4eq), DMF.

FIG. 27 shows the synthesis of trans-2-arylcyclopropylamines,trans-2-arylcyclopropyl-carboxyl acids and trans-2-arylethylepoxidylcarboxyl acids.

FIG. 28 shows the synthesis of alkene amino acids and alkyl epoxidylamino acids.

FIG. 29 shows the synthesis of hydrophilic Tut analogs for the synthesisof the hydrophilic prodrugs of antimitotic agents.

FIG. 30 shows the synthesis of the hydrophilic prodrugs of antimitoticagents for the conjugation with a cell binding agent.

FIG. 31 shows the synthesis of the hydrophilic prodrugs of antimitoticagents for the conjugation with an antibody.

FIG. 32 shows the synthesis of the hydrophilic prodrugs of antimitoticagents for the conjugation with an antibody.

FIG. 33 shows the in vitro cytotoxic effects of antiCD22antibody-antimitotic agent (TZ01˜TZ09) conjugates with drug/antibodyratio (DI A) 3.0-4.3 on Ramos (Burkitt lymphoma cell line). The cellswere incubated with the conjugates for 5 days and the IC₅₀ values areindicated on the figure.

FIG. 34 shows the cytotoxic effects of Trastuzumab-antimitotic agent(TZ03, TZ04, and TZ07) conjugates with drug/antibody ratio (DAR) 3.5-4.0on KPL-4 (breast cancer cell line). It also shows that Trastuzumab-TZ03conjugate induces a specifically potent antiproliferative effect—withIC₅₀=90 pM in the absence of unconjugated Trastuzumab and IC₅₀>20 nM inthe presence of 1 micromole concentration of Trastuzumab (to saturatethe antigen binding) respectively. The specificity window is >222(IC₅₀=20 nM/IC₅₀=0.09 nM).

FIG. 35 shows the cytotoxic effects of antiCD22 antibody-antimitoticagent (TZ03, TZ04, and TZ07) conjugates with drug/antibody ratio (D/A)3.8˜4.2 and of unconjugated CD22 antibody as well as of CD20 antibody(Rituximab) on BJAB (Burkitt lymphoma cell line). It shows that theconjugate induces much more potent antiproliferative effect—withIC₅₀=5˜19 pM than the unconjugated antibodies with IC₅₀>20 nM. Thespecificity windows when used 1 micromole concentration of unconjugatedCD22 antibody to saturate the antigen binding are >660 (IC₅₀=3.3nM/IC₅₀=0.005 nM) for huCD22-TZ03 conjugate and >790 (IC₅₀=15nM/IC₅₀=0.019 nM) for huCD-TZ07 conjugate.

FIGS. 36A-36J display the structure of the antimitotic drugs madethrough solid phase synthesis and their molecular ion of mass spectraand in vitro cytotoxicity of these drugs against Ramos cell (ATCC, ahuman Burkitt's lymphoma cell).

FIGS. 37A-37E display the structures of some experimentalantibody-antimitotic agent conjugates.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1 Definitions

“Alkyl” means an aliphatic hydrocarbon group which may be straight orbranched having 1 to 8 carbon atoms in the chain or cyclic. “Branched”means that one or more lower alkyl groups such as methyl, ethyl orpropyl are attached to a linear alkyl chain. Exemplary alkyl groupsinclude methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl,3-pentyl, octyl, nonyl, decyl, cyclopentyl, cyclohexyl,2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl,2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl,3-methylhexyl, 2,2-dimethylhexyl, 2,4-dimethylehexyl, 2,5-dimethylhexyl,3,5-dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl,n-heptyl, isoheptyl, n-octyl, and isooctyl. A C₁˜C₈ alkyl group can beunsubstituted or substituted with one or more groups including, but notlimited to, —C₁˜C₈ alkyl, —O—(C₁˜C₈ alkyl), —O—(C₁˜C₈ alkyl), -aryl,—C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′,—S(O)₂R′, —S(O)R′, —OH, -halogen (F, Cl, Br, or I), —N₃, —NH₂, —NH(R′),—N(R′)₂ and —CN; where each R′ is independently selected from —C₁˜C₈alkyl and aryl.

A “C₃˜C₈ carbocyle” means a 3-, 4-, 5-, 6-, 7- or 8-membered saturatedor unsaturated non-aromatic carbocyclic ring. Representative C₃˜C₈include, but are not limited to, -cyclopropyl, -cyclobutyl,-cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl,-1,3-cyclohexadienyl, 1,4-cyclohexadienyl, -cycloheptyl,-1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cycloheptatrienyl,-cyclooctyl, and -cyclooctadienyl. A C₃˜C₈ carbocycle group can beunsubstituted or substituted with one or more groups including, but notlimited to, —C₁˜C₈ alkyl, —O—(C₁˜C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′,C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′,—OH, -halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ isindependently selected from —C₁˜C₈ alkyl and aryl.

A “C₃˜C₈ carbocyclo” refers to a C₃˜C₈ carbocycle group defined abovewherein one of hydrogen atoms on the carbocyle is replaced with a bond.

“Alkenyl” means an aliphatic hydrocarbon group containing acarbon-carbon double bond and which may be straight or branched having 2to 8 carbon atoms in the chain. Exemplary alkenyl groups includeethenyl, propenyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl, n-pentenyl,hexylenyl, heptenyl, octenyl.

“Alkynyl” means an aliphatic hydrocarbon group containing acarbon-carbon triple bond and which may be straight or branched having 2to 8 carbon atoms in the chain. Exemplary alkynyl groups includeethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, pentynyl,n-pentynyl, hexylynyl, heptynyl, and octynyl.

“Heteroalkyl” is C₂˜C₈ alkyl in which one to four carbon atoms areindependently replaced with a heteroatom from the group consisting of O,S and N.

“Aryl” or Ar refers to an aromatic or hetero aromatic group, composed ofone or several rings, comprising three to fourteen carbon atoms,preferentially six to ten carbon atoms. The term of hetero aromaticgroup refers one or several carbon on aromatic group, preferentiallyone, two, three or four carbon atoms are replaced by O, N, Si, Se, P orS, preferentially by O, S, and N. The term aryl or Ar also refers to aaromatic group, wherein one or several H atoms are replacedindependently by R¹³, F, Cl, Br, I, OR¹³, or SR¹³, NR¹³R¹⁴, N═NR¹³,N═R¹³, NR¹³R¹⁴, NO₂, SOR¹³R¹⁴, SO₂R¹³, SO₃R¹³, OSO₃R¹³, PR¹³R¹⁴,POR¹³R¹⁴, PO₂R¹³R¹⁴, OPO₃R¹³R¹⁴, or PO₃R¹³R¹⁴ wherein R¹³, R¹⁴ areindependently H, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, arylalkyl,carbonyl, or pharmaceutical salts.

“Halogen atom” refers to fluorine, chlorine, bromine or iodine atom;preferably fluorine and chlorine atom.

“Heterocycle” refers to an aromatic or non-aromatic C₂˜C₈ heterocycle inwhich one to four of the ring carbon atoms are independently replacedwith a heteroatom from the group of O, N, S Se, and P. Preferableheteroatoms are oxygen, nitrogen and sulfur. Suitable heterocycles arealso disclosed in The Handbook of Chemistry and Physics, 78th Edition,CRC Press, Inc., 1997-1998, p. 2-25 to 2-26, the disclosure of which ishereby incorporated by reference. Preferred non aromatic heterocyclicinclude, but are not limited to epoxy, aziridinyl, thiiranyl,pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxiranyl,tetrahydrofuranyl, dioxolanyl, tetrahydro-pyranyl, dioxanyl, dioxolanyl,piperidyl, piperazinyl, morpholinyl, pyranyl, imidazolinyl, pyrrolinyl,pyrazolinyl, thiazolidinyl, tetrahydrothiopyranyl, dithianyl,thiomorpholinyl, dihydro-pyranyl, tetrahydropyranyl, dihydropyranyl,tetrahydro-pyridyl, dihydropyridyl, tetrahydropyrinidinyl,dihydrothiopyranyl, azepanyl, as well as the fused systems resultingfrom the condensation with a phenyl group.

The term “heteroaryl” or aromatic heterocycles refers to a 5 to 14,preferably 5 to 10 membered aromatic hetero, mono-, bi- or multicyclicring. Examples include pyrrolyl, pyridyl, pyrazolyl, thienyl,pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quinolinyl, purinyl,imidazolyl, thienyl, thiazolyl, benzothiazolyl, furanyl, benzofuranyl,1,2,4-thiadiazolyl, isothiazolyl, triazoyl, tetrazolyl, isoquinolyl,benzothienyl, isobenzofuryl, pyrazolyl, carbazolyl, benzimidazolyl,isoxazolyl, pyridyl-N-oxide, as well as the fused systems resulting fromthe condensation with a phenyl group.

“Alkyl”, “cycloalkyl”, “alkenyl”, “alkynyl”, “aryl”, “heteroaryl”,“heterocycle” and the like refer also to the corresponding “alkylene”,“cycloalkylene”, “alkenylene”, “alkynylene”, “arylene”, “heteroarylene”,“heterocyclene” and the likes which are formed by the removal of twohydrogen atoms.

“Pharmaceutically” or “pharmaceutically acceptable” refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, or a human, asappropriate.

“Pharmaceutically acceptable excipient” includes any carriers, diluents,adjuvants, or vehicles, such as preserving or antioxidant agents,fillers, disintegrating agents, wetting agents, emulsifying agents,suspending agents, solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, its use inthe therapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions as suitabletherapeutic combinations.

As used herein, “pharmaceutical salts” refer to derivatives of thedisclosed compounds wherein the parent compound is modified by makingacid or base salts thereof. The pharmaceutically acceptable saltsinclude the conventional non-toxic salts or the quaternary ammoniumsalts of the parent compound formed, for example, from non-toxicinorganic or organic acids. For example, such conventional non-toxicsalts include those derived from inorganic acids such as hydrochloric,hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; andthe salts prepared from organic acids such as acetic, propionic,succinic, tartaric, citric, methanesulfonic, benzenesulfonic,glucuronic, glutamic, benzoic, salicylic, toluenesulfonic, oxalic,fumaric, maleic, lactic and the like. Further addition salts includeammonium salts such as tromethamine, meglumine, epolamine, etc., metalsalts such as sodium, potassium, calcium, zinc or magnesium.

The pharmaceutical salts of the present invention can be synthesizedfrom the parent compound which contains a basic or acidic moiety byconventional chemical methods. Generally, such salts can be prepared byreaction of the free acid or base forms of these compounds with astoichiometric amount of the appropriate base or acid in water or in anorganic solvent, or in a mixture of the two. Generally, non-aqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, p. 1418, the disclosure of which is hereby incorporated byreference.

5.2 Drug-Linker-Binding Ligand Conjugates

As stated above, this invention provides a cell surface bindingmolecule-antimitotic agent conjugate of formula (I):

and pharmaceutical acceptable salts and solvates thereof

Wherein T is a targeting or binding ligand; L is a releasablelinker; - - - - - is a linkage bond that L connects to a molecule insidethe bracket independently; n is 1˜20 and m is 1˜10.

Inside the round bracket is a potent antimitotic agent/drug wherein R¹,R², R³, and R⁴ are independently C₁˜C₈ of alkyl, heteroalkyl; C₂˜C₈ ofheterocyclic, carbocyclic, alkylcycloalkyl, heterocycloalkyl, C₃˜C₈ ofaryl, Ar-alkyl, heteroalkylcycloalkyl, alkylcarbonyl; or two R′s, suchas R¹R², R²R³, R³R⁴, R⁵R⁶ and R¹²R¹³ can be 3-7 members of acarbocyclic, cycloalkyl, or heterocyclic, heterocycloalkyl ring system;Y is N or CH; In addition, R¹, R³, and R⁴ can be H; and R² can beabsent.

Wherein R⁵, R⁶, R⁸ and R¹⁰ are independently H, C₁˜C₄ of alkyl orheteroalkyl.

Wherein R⁷ is independently selected from H, R¹⁴, or —R¹⁴C(═O)X¹R¹⁵ or—R¹⁴X¹R¹⁵, wherein R¹⁴ and R¹⁵ are independently selected from C₁˜C₈ ofalkyl, or heteroalkyl; C₂˜C₈ of alkenyl, alkynyl; heterocyclic,carbocyclic, cycloalkyl; C₃˜C₈ of aryl, heterocycloalkyl, heteroaralkyl,heteroalkylcycloalkyl, alkylcarbonyl; X¹ is selected from O, S, S—S, NH,or NR¹⁴.

Wherein R⁹ is independently H, —O—, —OR¹⁴—, —OC(═O)R¹⁴—, —OC(═O)NHR¹⁴—,—OC(═O)R¹⁴SSR¹⁵—, OP(═O)(OR¹⁴)—, or OR¹⁴OP(═O)(OR¹⁵), wherein R¹⁴, R¹⁵are independently C₁˜C₈ of alkyl, heteroalkyl; C₂˜C₈ of alkenyl,alkynyl, heterocyclic, carbocyclic; C₃˜C₈ of aryl, cycloalkyl,alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl,alkylcarbonyl.

Wherein R¹ is independently H, R¹⁴, —R¹⁴C(═O)R¹⁶, —R¹⁴X²R¹⁶,—R¹⁴C(═O)X², wherein X² is —O—, —S—, —NH—, —N(R¹⁴)—, —O—R¹⁴—, —S—R¹⁴—,—S(═O)—R¹⁴—, or —NHR¹⁴—; R¹⁴ is C₁˜C₈ of alkyl, heteroalkyl; C₂˜C₈ ofalkenyl, alkynyl, heterocyclic, carbocyclic; C₃˜C₈ of aryl, cycloalkyl,alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl,alkylcarbonyl; R¹⁶ is H, OH, R¹⁴ or one to four amino acid units;

Wherein R¹² is independently R¹⁴, —O—, —S—, —N—, ═N—, ═NNH—, —NH(R¹⁴)—,—OR¹⁴—, —C(O)O—C(O)OR¹⁶—, C(O)NH—, C(O)NHR¹⁴, —SR¹⁴—, —S(═O)R¹⁴—,—P(═O)(OR¹⁶)—, —OP(═O)(OR¹⁶)—, CH₂OP(═O)(OR¹⁶)—, —C(O)OP(═O)(OR¹⁶)—,—SO₂R¹⁶—. R¹⁴ is independently C₁˜C₈ of alkyl, heteroalkyl; C₂˜C₈ ofalkenyl, alkynyl, hetero-cyclic, carbocyclic; C₃˜C₈ of aryl, cycloalkyl,alkylcycloalkyl, heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkyl,alkylcarbonyl. R¹⁶ is H, OH, R¹⁴ or one to four amino acid units;

Wherein R¹³ is C₁˜C₁₀ of alkyl, heteroalkyl, alkyl acid, alkyl amide,alkyl amine, or Ar; Ar refers to a aromatic or hetero aromatic group,composed of one or several rings, comprising four to ten carbon,preferentially four to six carbon atoms. The term of hetero aromaticgroup refers one or several carbon on aromatic group, preferentiallyone, two or three carbon atoms are replaced by O, N, Si, Se, P or S,preferentially O, S, N. The term aryl or Ar also refers to a aromaticgroup, wherein one or several H atoms are replaced independently by R¹⁷,F, Cl, Br, I, OR¹⁶, SR¹⁶, NR¹⁶R¹⁷, N═NR¹⁶, N═R¹⁶, NR¹⁶R¹⁷, NO₂,SOR¹⁶R¹⁷, SO₂R¹⁶, SO₃R¹⁶, OSO₃R¹⁶, PR¹⁶R¹⁷, POR¹⁶R¹⁷, PO₂R¹⁶R¹⁷,OP(O)(OR)₂, OCH₂OP(O)(OR¹⁷)₂, OC(O)OP(O)(OR¹⁷)₂, PO(OR¹⁶)(OR¹⁷),OP(O)(OR¹⁶)OP(O)(OR¹⁷)₂, OC(O)R¹⁷ or OC(O)NHR¹⁷, wherein R¹⁶, R¹⁷ areindependently H, C₁˜C₈ of alkyl, heteroalkyl; C₂˜C₈ of alkenyl, alkynyl,heterocyclic, carbocyclic; C₃˜C₈ of aryl, cycloalkyl, alkylcycloalkyl,heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl orC₄˜C₁₂ glycosides, or pharmaceutical salts.

In addition, R¹² can be H when R¹⁰ is not H, or when R¹³ is:

wherein Z¹ is H, CH₂OP(O)(OR⁸)₂, C(O)OP(O)(OR¹⁸)₂, PO(OR¹⁸)₂, C(O)R¹⁸,C(O)NHR¹⁸, SO₂(OR¹⁸), C₄˜C₁₂ glycosides or C₁˜C₈ of alkyl, carboxyalkyl,heterocyclic; R¹⁸ is H, C₁˜C₈ of alkyl, carboxyalkyl, heteroalkyl; C₂˜C₈of alkenyl, alkynyl, heterocyclic; C₃˜C₈ of aryl, alkylcarbonyl; R¹⁹ isH, OH, NH₂, OSO₂(OR⁸), XCH₂OP(O)(OR¹⁸)₂, XPO(OR¹⁸)₂, XC(O)R¹⁸,XC(O)NHR¹⁸, C₁˜C₈ of alkyl, carboxyalkyl, carboxylic acid derivative;C₂˜C₈ of alkenyl, alkynyl, heterocyclic; C₃˜C₈ of aryl, alkylcarbonyl;or pharmaceutical salts; X is O, S, NH; Y¹ and Y² are N or CHrespectively.

Or R¹² can be H when R¹¹ is:

wherein X² is O, S, N—R⁸; R⁸ is H, C₁˜C₆ of alkyl or heteroalkyl

In another embodiment, conjugates of antimitotic agents have the formula(II)

and pharmaceutical acceptable salts and solvates thereof

Wherein T is a targeting or binding ligand; L is a releasablelinker; - - - - - is a linkage bond that L connects to a molecule insidethe bracket independently; n is 1˜20 and m is 1˜10.

Inside the round bracket is a potent antimitotic agent/drug wherein R¹,R², R³, and R⁴ are independently C₁˜C₈ of alkyl, heteroalkyl; C₂˜C₈ ofheterocyclic, carbocyclic, alkylcycloalkyl, heterocycloalkyl, C₃˜C₈ ofaryl, Ar-alkyl, heteroalkylcycloalkyl, alkylcarbonyl; or two R's, suchas R¹R², R²R³, R³R⁴, R⁵R⁶ and R¹²R¹³ can be 3-7 members of acarbocyclic, cycloalkyl, or heterocyclic, heterocycloalkyl ring system;Y is N or CH; In addition, R¹, R³, and R⁴ can be H; and R² can beabsent.

Wherein R⁵, R⁶, R⁸ and R¹⁰ are independently H, C₁˜C₄ of alkyl orheteroalkyl.

Wherein R⁷ is independently selected from H, R¹⁴, or —R¹⁴C(═O)XR¹⁵ or—R¹⁴X¹R¹⁵, wherein R¹⁴ and R¹⁵ are independently selected from C₁˜C₈ ofalkyl, or heteroalkyl; C₂˜C₈ of alkenyl, alkynyl; heterocyclic,carbocyclic, cycloalkyl; C₃˜C₈ of aryl, heterocycloalkyl, heteroaralkyl,heteroalkylcycloalkyl, alkylcarbonyl; X¹ is selected from O, S, S—S, NH,or NR¹⁴.

Wherein R⁹ is independently H, —O—, —OR¹⁴, —OC(═O)R¹⁴, —OC(═O)NHR¹⁴,—OC(═O)R¹⁴SSR¹⁵, OP(═O)(OR¹⁴), or OR¹⁴OP(═O)(OR¹⁵), wherein R¹⁴, R¹⁵ areindependently C₁˜C₈ of alkyl, heteroalkyl; C₂˜C₈ of alkenyl, alkynyl,heterocyclic, carbocyclic; C₃˜C₈ of aryl, cycloalkyl, alkylcycloalkyl,heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl.

Wherein R¹¹ is independently H, R¹⁴, —R¹⁴C(═O)R¹⁶, —R¹⁴X²R¹⁶,—R⁴C(═O)X², wherein X² is —O—, —S—, —NH—, —N(R¹⁴)—, —O—R¹⁴—, —S—R¹⁴—,—S(═O)—R¹⁴—, or —NHR¹⁴—; R¹⁴ is C₁˜C₈ of alkyl, heteroalkyl; C₂˜C₈ ofalkenyl, alkynyl, heterocyclic, carbocyclic; C₃˜C₈ of aryl, cycloalkyl,alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl,alkylcarbonyl; R¹⁶ is H, OH, R¹⁴ or one to four amino acid units;

Wherein R¹² is independently R¹⁴, —OH, —SH, —NH₂, ═NH, ═NNH₂, —NH(R¹⁴),—OR¹⁴, COR¹⁶, COOR¹⁴, C(O)NH₂, C(O)NHR¹⁴, —SR¹⁴, —S(═O)R¹⁴,—P(═O)(OR¹⁶)₂, —OP(═O)(OR¹⁶)₂, —CH₂OP(═O)(OR¹⁶)₂, —SO₂R¹⁶. R¹⁴ isindependently C₁˜C₈ of alkyl, heteroalkyl; C₂˜C₈ of alkenyl, alkynyl,hetero-cyclic, carbocyclic; C₃˜C₈ of aryl, cycloalkyl, alkylcycloalkyl,heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkyl, alkylcarbonyl.R¹⁶ is H, OH, R¹⁴ or one to four amino acid units;

Wherein R¹³ is C₁˜C₁₀ of alkyl, heteroalkyl, alkyl acid, alkyl amide,alkyl amine, or Ar; Ar refers to a aromatic or hetero aromatic group,composed of one or several rings, comprising four to ten carbon,preferentially four to six carbon atoms. The term of hetero aromaticgroup refers one or several carbon on aromatic group, preferentiallyone, two or three carbon atoms are replaced by O, N, Si, Se, P or S,preferentially O, S, N. The term aryl or Ar also refers to a aromaticgroup, wherein one or several H atoms are replaced independently by R¹⁷,F, Cl, Br, I, OR¹⁶, SR¹⁶, NR¹⁶R¹⁷, N═NR¹⁶, N═R¹⁶, NR¹⁶R¹⁷, NO₂,SOR¹⁶R¹⁷, SO₂R¹⁶, SO₃R¹⁶, OSO₃R¹⁶, PR¹⁶R¹⁷, POR¹⁶R¹⁷, PO₂R¹⁶R¹⁷,OP(O)(OR)₂, OCH₂OP(O)(OR⁷)₂, OC(O)OP(O)(OR⁷)₂, PO(OR¹⁶)(OR¹⁷),OPO(OR¹⁶)OPO(OR¹⁶)(OR¹⁷), OC(O)R¹⁷ or OC(O)NHR¹⁷, wherein R¹⁶, R¹⁷ areindependently H, C₁˜C₈ of alkyl, heteroalkyl; C₂˜C₈ of alkenyl, alkynyl,heterocyclic, carbocyclic; C₃˜C₈ of aryl, cycloalkyl, alkylcycloalkyl,heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl orC₄˜C₁₂ glycosides, or pharmaceutical salts.

In addition, R¹² can be H when R¹⁰ is not H, or when R¹³ is:

wherein Z¹ is H, CH₂OP(O)(OR¹⁸)₂, C(O)OP(O)(OR¹⁸)₂, PO(OR¹⁸)₂, C(O)R¹⁸,C(O)NHR¹⁸, SO₂(OR¹⁸), C₄˜C₁₂ glycosides or C₁˜C₈ of alkyl, carboxyalkyl,heterocyclic; R¹⁸ is H, C₁˜C₈ of alkyl, carboxyalkyl, heteroalkyl; C₂˜C₈of alkenyl, alkynyl, heterocyclic; C₃˜C₈ of aryl, alkylcarbonyl; R¹⁹ isH, OH, NH₂, OSO₂(OR¹⁸), XCH₂OP(O)(OR¹⁸)₂, XC(O)OP(O)(OR¹⁸)₂, XPO(OR¹⁸)₂,XC(O)R¹⁸, XC(O)NHR¹⁸, C₁˜C₈ of alkyl, carboxyalkyl, carboxylic acidderivative; C₂˜C₈ of alkenyl, alkynyl, heterocyclic; C₃˜C₈ of aryl,alkylcarbonyl; or pharmaceutical salts; X is O, S, NH; Y¹ and Y² are Nor CH respectively.Or R¹² can be H when R¹¹ is:

X² is O, S, N—R⁸; R⁸ is H, C₁˜C₆ of alkyl or heteroalkyl

Illustrative classes of compounds of formula II have the structures:

wherein Aa is a natural or an unnatural amino acid; n is 1˜20; q=1˜5;X′, Y′ and Z′ are independently CH, O, S, NH, or NR²²; R²² and R²³ areindependently C₁˜C₈ of alkyl; C₂˜C₈ of alkenyl, alkynyl, heteroalkyl;C₃˜C₈ of aryl, heterocyclic, carbocyclic, cycloalkyl, alkylcycloalkyl,heterocycloalkyl, Ar-alkyl, heteroalkylcycloalkyl, heteroaralkyl, or—(OCH₂CH₂)_(n)—; R′ and R″ are independently H or CH₃. Inside the roundbrackets are the antimitotic drugs and the inside square brackets arethe antimitotic drug with linkers.

In another embodiment, a conjugate of a cell bindingmolecule-antimitotic agent has the formula (III):

wherein T, L, m, Y, R¹, R², R³, R⁴, R⁵, R⁶, R₈, R⁹, R¹⁰, R¹¹, R¹², R¹³and n are defined the same as in formula (II).

Wherein R⁷ is independently selected from R¹⁴, or —R¹⁴C(═O)X¹R¹⁵— or—R¹⁴XR¹⁵—. R¹⁴ and R¹⁵ are independently selected from C₁˜C₈ of alkyl,or heteroalkyl; C₂˜C₈ of alkenyl, alkynyl; heterocyclic, carbocyclic,cycloalkyl; C₃˜C₈ of aryl, heterocycloalkyl, heteroaralkyl,heteroalkylcycloalkyl, alkylcarbonyl; X¹ is selected from O, S, S—S, NH,or NR¹⁴.

Illustrative examples of compounds of formula (III) have the structures:

wherein Ar, n, q, X′, Y′, Z′, R²², R²³, R′ and R″ are defined the sameas for formula IIa˜IIr.

In another embodiment, a binding molecule-antimitotic agent conjugatehas the formula (IV)

wherein T, L, m, Y, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R, R¹², R¹³ and nare defined the same as in formula (II).

Wherein R⁹ is independently, —O—, —OR¹⁴—, —OC(═O)R¹⁴—, —OC(═O)NHR¹⁴—,—OC(═O)R¹⁴SSR¹⁵—, —OP(═O)(OR¹⁴)O—, wherein R¹⁴, R¹⁵ are independentlyC₁˜C₈ of alkyl, heteroalkyl; C₃˜C₈ of aryl, heteroaryl, heterocyclic,carbocyclic, cycloalkyl, alkylcycloalkyl, heterocycloalkyl,heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl or pharmaceuticalsalts. In addition, R⁹ can be absent.

Illustrative examples of compounds of formula (IV) have the structures:

wherein Ar, Aa, n, q, X′, Y′, R²², R²³, R′ and R″ are defined the sameas for formula IIa˜IIr.

In another embodiment, a binding molecule-antimitotic agent conjugatehas the formula (V)

wherein T, L, m, Y, R¹, R², R³, R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹²,R¹³ and n are defined the same as in formula (II).

Wherein R¹¹ is independently —R¹⁴—, —R¹⁴C(═O)R¹⁷—, —R¹⁴X²R¹⁷—,—R¹⁴C(═O)X²—, wherein R¹⁷ is independently H, OH, C₁˜C₈ of alkyl; C₂˜C₈of alkenyl, alkynyl, heteroalkyl; C₃˜C₈ of aryl, arylene, heterocyclic,carbocyclic, heterocycloalkyl; or an amino acid, or two amino acidunits; X² is —O—, —S—, —NH—, —N(R¹⁴)—, —O—R¹⁴—, —S—R¹⁴—, —S(═O)—R¹⁴—, or—NHR¹⁴—; R¹⁴ is C₁˜C₈ of alkyl, heteroalkyl; C₂˜C₈ of alkenyl, alkynyl;C₃˜C₈ of aryl, heterocyclic, carbocyclic, cycloalkyl, alkylcycloalkyl,heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl.

Illustrative examples of compounds of formula (V) have the structures:

Wherein Ar, Aa, n, q, X′, R²², R²³, R′, and R″ are defined the same asfor formula IIa˜IIr.

In another embodiment, a conjugates of a cell binding-antimitotic agenthave the formula (VI)

wherein T, L, m, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹¹, R¹³ and n aredefined the same as formula (II).

Wherein R¹² is independently R¹⁴, —O—, —S—, —Ni—, ═N—, ═NNH—, —N(R⁴)—,—OR⁴—, C(O)O—, C(O)NH—, C(O)NR—, —SR¹⁴—, —S(═O)R¹⁴—, —NHR¹⁴—,—CH₂OP(═O)(OR¹⁵)—, —P(═O)(OR¹⁵)—, —OP(═O)(OR¹⁵)O—, —SO₂R¹⁴. R¹⁴, R¹⁵ areindependently C₁˜C₂ of alkyl, heteroalkyl; C₂˜C₈ of alkenyl, alkynyl;C₃˜C₈ of aryl, heterocyclic, carbocyclic, cycloalkyl, alkylcycloalkyl,heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkyl, alkylcarbonyl.

Illustrative examples of compounds of formula VI have the followingstructures:

Wherein Ar, Aa, n, q, X′, R²², R²³, R′ and R″ are defined the same asfor formula IIa˜IIr.

In another embodiment, the conjugates of the cell-surface bindingmolecule-antimitotic agents have the formula (VII)

Wherein T, L, m, R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₁, R₁₂ and n aredefined the same as in formula (II).

Wherein R¹³ is C₁˜C₁₀ of alkyl, heteroalkyl, alkyl acid, alkyl amide,alkyl amine, or Ar; Ar refers to a aromatic or hetero aromatic group,composed of one or several rings, comprising four to ten carbon,preferentially four to six carbon atoms. The term of hetero aromaticgroup refers one or several carbon on aromatic group, preferentiallyone, two or three carbon atoms are replaced by O, N, Si, Se, P or S,preferentially O, S, N. The term aryl or Ar also refers to a aromaticgroup, wherein one or several H atoms are replaced independently by R¹⁸,F, Cl, Br, I, OR¹⁶, SR¹⁶, R¹⁶R¹⁸, N═NR¹⁶, N═R¹⁶, R¹⁶R¹⁸, NO₂, SOR¹⁶R¹⁸,SO₂R¹⁶, SO₂R¹⁶, SO₃R¹⁶, OSO₃R¹⁶, PR¹⁶R¹⁸, POR¹⁶R¹⁸, PO₂R¹⁶R¹⁸,OPO₃R¹⁶R¹⁸, OPO₂R¹⁶OPO₃R¹⁸ or PO₃R¹⁶R¹⁸ wherein R¹⁶, R¹⁸ areindependently H, C₁˜C₈ of alkyl; C₂˜C₈ of alkenyl, alkynyl, heteroalkyl;C₃˜C₈ of aryl, heterocyclic, carbocyclic, cycloalkyl, alkylcycloalkyl,heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkyl, alkylcarbonyl;or C₄˜C₁₂ glycosides; or pharmaceutical salts.

In addition, R¹² can be H when R¹⁰ is not H, or when R¹³ is:

wherein Z¹ is H, CH₂OP(O)(OR¹⁸)₂, C(O)OP(O)(OR¹⁸)₂, PO(OR¹⁸)₂,PO(OR⁸)OPO(OR¹⁸)₂, C(O)R⁸, C(O)NHR¹⁸, SO₂(OR⁸), C₄˜C₁₂ glycosides, orC₁˜C₈ of alkyl, carboxyalkyl, heterocyclic; R¹⁸ is H, C₁˜C₈ of alkyl,carboxyalkyl, heteroalkyl; C₂˜C₈ of alkenyl, alkynyl, heterocyclic;C₃˜C₈ of aryl, alkylcarbonyl; R¹⁹ is H, OH, NH₂, OSO₂(OR¹⁸),XCH₂OP(O)(OR¹⁸)₂, XC(O)OP(O)(OR¹⁸)₂, XPO(OR¹⁸)₂, XC(O)R¹⁸, XC(O)NHR¹⁸,C₁˜C₈ of alkyl, carboxyalkyl, carboxylic acid derivative; C₂˜C₈ ofalkenyl, alkynyl, heterocyclic; C₃˜C₈ of aryl, alkylcarbonyl; orpharmaceutical salts; X is O, S, NH; Y¹ and Y² are N or CH respectively.

Or R¹² can be H when R¹¹ is:

X² is O, S, N—R⁸; R⁸ is H, C₁˜C₄ of alkyl or heteroalkyl.

Illustrative examples of compounds of formula VII have the followingstructures:

Wherein Ar, Aa, n, q, X′, R²², R²³, R′ and R″ are defined the same asfor formula IIa˜IIr; R²⁴ is H or CH₃.

In another embodiment, the synthetic routes to produce the antimitoticagents and their conjugation to a cell-surface receptor bindingmolecules of the present invention are exampled, but not limited to,shown in FIGS. 1-32.

In another embodiment, the releasable linker (L) used for theconjugation of the present invention is a chain of atoms selected fromC, N, O, S, Si, and P that covalently connects the cell-surface bindingligand (T) to the potent antimitotic agents. The linker may have a widevariety of lengths, such as in the range from about 2 to about 100atoms, The atoms used in forming the linker may be combined in allchemically relevant ways, such as forming alkylene, alkenylene, andalkynylene, ethers, polyoxyalkylene, esters, amines, imines, polyamines,hydrazines, hydrazones, amides, ureas, semicarbazides, carbazides,alkoxyamines, alkoxylamines, urethanes, amino acids, acyloxylamines,hydroxamic acids, and many others. In addition, it is to be understoodthat the atoms forming the releasable linker (L) may be either saturatedor unsaturated, or may be radicals, or may be cyclized upon each otherto form divalent cyclic structures, including cyclo alkanes, cyclicethers, cyclic amines, arylenes, heteroarylenes, and the like in thelinker.

The term releasable linker refers to a linker that includes at least onebond that can be broken under physiological conditions, such as apH-labile, acid-labile, base-labile, oxidatively labile, metabolicallylabile, biochemically labile, or enzyme-labile bond. It is appreciatedthat such physiological conditions resulting in bond breaking do notnecessarily include a biological or metabolic process, and instead mayinclude a standard chemical reaction, such as a hydrolysis orsubstitution reaction, for example, an endosome having a lower pH thancytosolic pH, and/or disulfide bond exchange reaction with aintracellular thiol, such as a millimolar range of abundant ofglutathione inside the malignant cells.

The releasable linker L of conjugates may have the formula:-Ww-(Aa)r-Vv- wherein: -W- is a Stretcher unit; w is 0 or 1; each -Aa-is independently an Amino Acid unit; r is independently an integerranging from 0 to 12; -V- is a Spacer unit; and v is 0, 1 or 2.

The Stretcher unit (-W-), when present, links a targeted bindingmolecular unit (T) to an amino acid unit (-Aa-), or links V when an Aais not present. The Stretcher unit W may independently contain aself-immolative spacer, peptidyl units, a hydrazone bond, disulfide orthioether bonds. In this regard a binding molecular (T) has a functionalgroup that can form a bond with a functional group of a Stretcher.Useful functional groups that can be present on a binding molecular,either naturally or via chemical manipulation include, but are notlimited to, sulfhydryl (—SH), amino, hydroxyl, carbonyl, the anomerichydroxyl group of a carbohydrate, and carboxyl. Preferred functionalgroups are sulfhydryl, carboxy and amino. Sulfhydryl groups can begenerated by reduction of an intramolecular disulfide bond of a bindingligand, such as of an antibody. Alternatively, sulfhydryl groups can begenerated by reaction of an amino group of a lysine moiety of a bindingmolecular using 2-iminothiolane (Traut's reagent) or thiolactone oranother sulfhydryl generating reagent, such as modifies T with adisulfide bond linker, or a thiol ester following by reduction orhydrolysis respectively.

Illustrative examples of W linked to T have the structures:

wherein R²⁰ and R²¹ are selected from —C₁˜C₉ alkylene-, —C₁˜C₇carbocyclo-, —O—(C₁˜C₈ alkyl)-, -arylene-, —C₁˜C₉ alkylene-arylene-,-arylene, —C₁˜C₉ alkylene-, —C₁˜C₉ alkylene-(C₁˜C₈ carbocyclo)-, —(C₃˜C₇carbocyclo)-C₁˜C₉ alkylene-, —C₃˜C₈ heterocyclo-, —C₁-C₁₀alkylene-(C₃˜C₈ heterocyclo)-, —(C₃˜C₈ heterocyclo)-C₁˜C₉ alkylene-,—(CH₂CH₂O)_(k)—, —(CH(CH₃)CH₂O)_(k)—, and —(CH₂CH₂O)_(k)—CH₂—; k is aninteger ranging from 1-20.; R′ and R″ are independently H or CH₃.

In another embodiment, conjugation of W to T covalently as illustratedabove can be via various chemical reactions.

Examples of the formation of amide linkages:

Wherein the Stretcher unit contains a reactive site of E, which can forman amide bond with a primary or secondary amino group of a bindingmolecule. Example of the reactive E, includes, but is not limited to,such as hydroxysuccinimidyl esters (NHS, Sulfa-NHS, etc), 4-nitrophenylesters, pentafluorophenyl esters, tetrafluorophenyl (includessulfo-tetrafluorophenyl) esters, anhydrides, acid chlorides, sulfonylchlorides, isocyanates and isothiocyanates.

Examples of thiol ether or disulfide bond linkages:

Wherein the Stretcher unit contains a sulfhydryl reactive site, whichcan form a thiol ether or disulfide bond with a thiol group which isgenerated by reduction of an intramolecular disulfide bond of thebinding ligand T, or generated by a chemical modification on the bindingligand T.

In yet another aspect of the invention, the reactive group of theStretcher contains a reactive site that is reactive to an aldehyde(—CHO) or a ketone (—C(═O)R) group that can be chemically modified on abinding molecular T. For example, a carbohydrate on a binding molecularT can be mildly oxidized using a reagent such as sodium periodate togenerate an aldehyde or a ketone (—C(═O)R) group; or an amine on anamino acid at the N-termini of antibodies (or proteins or peptides) canreact with pyridoxal 5′-phosphate (PLP) in a buffer solution tointroduce ketone groups. The resulting (—C═O) unit can be condensed witha Stretcher that contains a functionality such as a hydrazide, an oxime,a primary or secondary amine, a hydrazine, a thiosemicarbazone, ahydrazine carboxylate, and an arylhydrazide.

Examples of the conjugation of the hydrazone, or the oxime or iminelinkages:

wherein R²⁰ and R²¹ are described above, R²⁵ is an organic substituentof an amino acid.

In another aspect of the invention, the Stretchers (which may contain aspacer V and/or an amino acid) can be linked to the binding molecules(T), followed by conjugation of a potent antimitotic agent to thebinding molecule-stretcher moiety in an aqueous buffered solution.

Examples of these two-step conjugations (a cytotoxic drug linked to R¹⁶is omitted here) are:

wherein E includes, but is not limited to, such as hydroxysuccinimidylesters (NHS, SulfoNHS, etc), 4-nitrophenyl esters, pentafluorophenylesters, tetrafluorophenyl (includes sulfo-tetrafluorophenyl) esters,anhydrides, acid chlorides, sulfonyl chlorides, isocyanates andisothiocyanates. R′ and R″ are independently H or CH₃; R²⁰, R¹⁶ and Arare defined in various embodiment throughout this inventions; R²⁶ is H,or F, or NO₂ independently; J is F, Cl, Br, I, tosylate (TsO) ormesylate (MsO) independently and wherein

bears at least one antimitotic agent/drug as

In another aspect of the invention, the Stretchers can be linked to apotent antimitotic agent first, followed by conjugation of the bindingmolecules (T) in an aqueous pH 3˜10 (preferably pH 5˜8.5) bufferedsolution containing up to 50% of organic cosolvents. Examples of thesekinds of two-step conjugations:

wherein E includes, but is not limited to, such as hydroxysuccinimidylesters (NHS, SulfoNHS, etc), 4-nitrophenyl esters, pentafluorophenylesters, tetrafluorophenyl (includes sulfo-tetrafluorophenyl) esters,anhydrides, acid chlorides, sulfonyl chlorides, isocyanates andisothiocyanates. R′ and R″ are independently H or CH₃; R¹⁶, R²⁰ and Arare defined in various embodiment throughout this inventions; R²⁶ is H,or F, or NO₂ independently; J is F, Cl, Br, I, tosylate (TsO) ormesylate (MsO) independently and wherein

bears at least one antimitotic agent/drug.

The Amino Acid unit (-Aa-), when present, links the Stretcher unit tothe Spacer unit if the Spacer unit is present, links the Stretcher unitto the antimitotic agent unit if the Spacer unit is absent, and linksthe binding molecule (T) unit to the antimitotic agent unit if theStretcher unit and Spacer unit are absent. -(Aa)r- is a natural orunnatural amino acid, dipeptide, tripeptide, tetrapeptide, pentapeptide,hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide,undecapeptide or dodecapeptide unit, and r is an integer ranging from 0to 12. The term amino acid as used herein refers generally toaminoalkylcarboxylate, where the alkyl radical is optionallysubstituted, such as with alkyl, acyl, hydroxy alkyl, sulfhydrylalkyl,aminoalkyl, carboxyalkyl, and the like, The structures of the naturaland unnatural amino acids and peptides are described in the book: G. C.Barrett and D. T. Elmore, “Amino Acid and Peptide”, Cambridge UniversityPress, 2004. In addition, amino acid refers to beta, gamma, and longeramino acids with intra chain containing methyl, benzyl, hydroxymethyl,thiomethyl, carboxyl, carboxylmethyl, guanidinopropyl, and the like.More preferably the amino acid is selected from arginine, asparagine,aspartic acid, citrulline, cysteine, glycine, glutamic acid, leucine,lysine, glutamic acid, glutamine, serine, ornithine, phenylalanine,threonine, tyrosine, valine and the like.

The Amino Acid unit used in this invention can be enzymatically cleavedby one or more enzymes, including a tumor-associated protease, toliberate the antimitotic agent, which in one embodiment is protonated invivo upon release to provide an antimitotic agent.

The Spacer unit (-V-), when present, links an Amino Acid unit to theantimitotic agent when an Amino Acid unit is present. Alternately, theSpacer unit links the Stretcher unit to antimitotic agent when the AminoAcid unit is absent. The Spacer unit also links antimitotic agent to thebinding molecule (T) when both the Amino Acid unit and Stretcher unitare absent. The spacer linkers may contain function groups thatsubstantially increase the water solubility, biological transport,preferential renal clearance, uptake, absorption, biodistribution,and/or bioavailability of the conjugate are described herein. Spacerunits are of two general types: self-immolative and non self-immolative.A non self-immolative Spacer unit is one in which part or all of theSpacer unit remains bound to antimitotic agent after cleavage,particularly enzymatic, of an Amino Acid unit from the antimitoticagent-Linker-binding molecule conjugate or the antimitotic agent-LinkerCompound.

Examples of the self-immolative spacer linkers:

wherein the (*) atom is the point of attachment of additional spacer orreleasable linker units, the antimitotic agent, and/or the bindingmolecule (T); X, Y and Z³ are independently NH, O, or S; Z² is H, NH, Oor S independently. v is 0 or 1; Q in independently H, OH, C₁˜C₆ alkyl,(OCH₂CH₂)_(n)F, Cl, Br, I, OR¹⁷, or SR¹⁷, NR¹⁷R¹⁸, N═NR¹⁷, N═R¹⁷,NR¹⁷R¹⁸, NO₂, SOR¹⁷R¹⁸, SO₂R¹⁷, SO₃R¹⁷, OSO₃R¹⁷, PR¹⁷R¹⁸, POR¹⁷R¹⁸,PO₂R¹⁷R¹⁸, OPO(OR¹⁷)(OR¹⁸), OC(O)PO(OR¹⁷(OR⁸) or OCH₂PO(OR¹⁷(OR¹⁸)wherein R¹⁷, R¹⁸ are independently H, C₁˜C₈ of alkyl; C₂˜C₈ of alkenyl,alkynyl, heteroalkyl; C₃˜C₈ of aryl, heterocyclic, carbocyclic,cycloalkyl, heterocycloalkyl, heteroaralkyl, alkylcarbonyl; orpharmaceutical cation salts.

Examples of the non-self-immolative spacer linkers:

Where the (*) atom is the point of attachment of additional spacer orreleasable linkers, the antimitotic agents, and/or the bindingmolecules; m is 1˜10; n is 1˜20.

The binding molecule (T) may be of any kind presently known, or thatbecome known, molecule that binds to, complexes with or reacts with amoiety of a cell population sought to be therapeutically or otherwisebiologically modified. The binding molecule unit acts to deliver theantimitotic agents to the particular target cell population with whichthe binding molecule (T) reacts.

The cell binding agents include, but are not limited to, large molecularweight proteins such as, for example, full-length antibodies (polyclonaland monoclonal antibodies); single chain antibodies; fragments ofantibodies such as Fab, Fab′, F(ab′)₂, F_(v), [Parham, J. Immunol. 131,2895-2902 (1983)], fragments produced by a Fab expression library,anti-idiotypic (anti-Id) antibodies, CD R's, and epitope-bindingfragments of any of the above which immuno-specifically bind to cancercell antigens, viral antigens or microbial antigens; interferons (suchas type I, II, III); peptides; lymphokines such as IL-2, IL-3, IL-4,IL-6, GM-CSF, interferon-gamma (IFN-γ); hormones such as insulin, TRH(thyrotropin releasing hormones), MSH (melanocyte-stimulating hormone),steroid hormones, such as androgens and estrogens,melanocyte-stimulating hormone (MSH); growth factors andcolony-stimulating factors such as epidermal growth factors (EGF),granulocyte-macrophage colony-stimulating factor (GM-CSF), transforminggrowth factors (TGF), such as TGFα, TGFβ, insulin and insulin likegrowth factors (IGF-I, IGF-II) G-CSF, M-CSF and GM-CSF [Burgess,Immunology Today, 5, 155-158 (1984)]; vaccinia growth factors (VGF);fibroblast growth factors (FGFs); smaller molecular weight proteins,poly-peptide, peptides and peptide hormones, such as bombesin, gastrin,gastrin-releasing peptide; platelet-derived growth factors; interleukinand cytokines, such as interleukin-2 (IL-2), interleukin-6 (IL-6),leukemia inhibitory factors, granulocyte-macrophage colony-stimulatingfactor (GM-CSF); vitamins, such as folate; apoproteins andglycoproteins, such as transferrin {O'Keefe et al, 260 J. Biol. Chem.932-937 (1985)}; sugar-binding proteins or lipoproteins, such aslectins; cell nutrient-transport molecules; and small molecularinhibitors, such as prostate-specific membrane antigen (PSMA) inhibitorsand small molecular tyrosine kinase inhibitors (TKI), non-peptides orany other cell binding molecule or substance, such as bioactive polymers(Dhar, et al, Proc. Natl. Acad. Sci. 2008, 105, 17356-61); dendrimers(Lee, et al, Nat. Biotechnol. 2005, 23, 1517-26; Almutairi, et al; Proc.Natl. Acad. Sci. 2009, 106, 685-90); nanoparticles (Liang, et al, ACSNano, 2008, 19, 1309-12; Medarova, et al, Nat. Med. 2007, 13, 372-7;Javier, et al, Bioconjugate Chem. 2008, 19, 1309-12); liposomes(Medinai, et al, Curr. Phar. Des. 2004, 10, 2981-9); viral capsides(Flenniken, et al, Viruses Nanotechnol. 2009, 327, 71-93). In generalmonoclonal antibodies are preferred as a cell-surface binding agent ifan appropriate one is available.

Prior to conjugating with the antimitotic agents of this invention, thebinding molecules can be modified through attachment of a more specificpeptide, a protein, or a drug, or the other functional molecules with aheterobifunctional cross linker such as with linkers ofAmine-to-Nonselective (succinimidyl (NHS)-diazirine (SDA), NHSester/Azide), Amine-to-Sulfhydryl (NHS ester/maleimide, NHSester/pyridyldithiol, NHS esters/haloacetyl), Sulfhydryl-to-Carbohydrate(Maleimide/Hydrazide, Pyridyldithiol/Hydrazide), Hydroxyl-to-Sulfhydryl(Isocyanate/Maleimide), Amine-to-DNA (NHS ester/Psoralen),Amine-to-Carboxyl (Carbodiimide).

In the SDA linkage modification, the NHS ester of a SDA linker reactswith primary an amine group of a binding molecule backbone in pH 6-9buffer to form a stable amide bond upon release of NHS. Thenphotoactivation of the diarzirine with long-wave UV light (330-370 nm)creates a reactive carbene intermediate that can react with an aminegroup of a more specific peptide or a protein or the other functionalmolecule. The order of these two steps can be different as this: anamine group of a functional molecule reacts with a SDA linker firstfollowing by photoactive reaction of a binding molecule with long-waveUV light (330-370 nm). The SDA crosslinkers can be cleavable (with adisulfide bond inside such as SDAD linker).

In the NHS ester/Azide linkage modification, the NHS ester of the linkerreacts with primary an amine group of a binding molecule backbone in pH6-9 buffer to form a stable amide. Then an alkynyl group on a morespecific peptide or a protein or the other functional molecule reacts tothe azide on the other side of the linker via Azide-Alkyne HuisgenCycloaddition to form a 1,2,3-triazole linkage (click chemistry). Also,the NHS ester of the linker reacts with primary an amine group of afunctional molecule in pH 6-9 buffer to form a stable amide. Then analkynyl group being linked on a binding molecule reacts to the azide onthe other side of the linker via Azide-Alkyne Huisgen Cycloaddition toform a 1,2,3-triazole linkage.

In the Amine-to-Sulfhydryl linkage modification, the NHS ester of thelinker reacts with a primary amine group of a binding molecule backbonein pH 6-9 buffer to form a stable amide bond. Then a sulfhydryl on amore specific peptide or a protein or the other functional moleculereacts to the maleimide, or pyridyldithiol, or haloacetyl on the otherside of the Amine-tosulfhydryl linker at pH 4.5-8.5 to form a thioetheror a disulfide bond. The conjugation with the Amine-to-Sulfhydryl linkercan be in different orders. For instance, an amine group of a functionalmolecule can be reacted with the linker to form an amide bond first,following by reaction with a sulfhydryl on a binding molecule. Also asulfhydryl group of a functional molecule can be reacted with the linkerto form a thioether or a disulfide bond at pH 4.5-7 first, following byreaction with an amine group on a binding molecule at pH 6-9 to form anamide bond.

In the Sulfhydryl-to-Carbohydrate linkage modification, the sulfhydrylgroup of a binding molecule can be reacted with the maleimide or thepyridyldithiol on the linker to form a thioether or a disulfide bond atpH 4.5-8 first, Then a carbonyl (aldehyde/ketone) group on a functionalmolecule reacts with the hydrazide to form an hydrazone bond. Also thesulfhydryl group on a functional molecule can react with the linker toform a thioether or a disulfide bond at pH 4.5-8 first, following byreaction with a carbohydrate, or an oxidized carbohydrate, or ancarbonyl (aldehyde/ketone) group on a binding molecule form an hydrazonebond.

In the Hydroxyl-to-Sulfhydryl linkage modification, the sulfhydryl groupof a binding molecule can be reacted with the maleimide or thepyridyldithiol on the linker to form a thioether or a disulfide bond atpH 6˜8 first, Then a hydroxy group on a functional molecule reacts withthe isocyanate on the linker to form a carbamate bond at pH 8˜9. Alsothe sulfhydryl group on a functional molecule can react with the linkerto form a thioether or a disulfide bond at pH 6˜8 first, following byreaction with a hydroxy on a binding molecule form a carbamate bond atpH 8˜9.

In yet another aspect of the invention, the production of antibodiesused in the present invention involves in vivo or in vitro procedures orcombinations thereof. Methods for producing polyclonal anti-receptorpeptide antibodies are well-known in the art, such as in U.S. Pat. No.4,493,795 (to Nestor et al). A monoclonal antibody is typically made byfusing myeloma cells with the spleen cells from a mouse that has beenimmunized with the desired antigen (Kohler, G.; Milstein, C. (1975).Nature 256: 495-497). The detailed procedures are described in“Antibodies—A Laboratory Manual”, Harlow and Lane, eds., Cold SpringHarbor Laboratory Press, New York (1988), which is incorporated hereinby reference. Particularly monoclonal antibodies are produced byimmunizing mice, rats, hamsters or any other mammal with the antigen ofinterest such as the intact target cell, antigens isolated from thetarget cell, whole virus, attenuated whole virus, and viral proteins.Splenocytes are typically fused with myeloma cells using polyethyleneglycol (PEG) 6000. Fused hybrids are selected by their sensitivity toHAT (hypoxanthine-aminopterin-thymine). Hybridomas producing amonoclonal antibody useful in practicing this invention are identifiedby their ability to immunoreact specified receptors or inhibit receptoractivity on target cells.

A monoclonal antibody used in the present invention can be produced byinitiating a monoclonal hybridoma culture comprising a nutrient mediumcontaining a hybridoma that secretes antibody molecules of theappropriate antigen specificity. The culture is maintained underconditions and for a time period sufficient for the hybridoma to secretethe antibody molecules into the medium. The antibody-containing mediumis then collected. The antibody molecules can then be further isolatedby well-known techniques, such as using protein-A affinitychromatography; anion, cation, hydrophobic, or size exclusivechromatographies (particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography); centrifugation,differential solubility, or by any other standard technique for thepurification of proteins.

Media useful for the preparation of these compositions are bothwell-known in the art and commercially available and include syntheticculture media. An exemplary synthetic medium is Dulbecco's minimalessential medium (DMEM; Dulbecco et al., Viral. 8:396 (1959))supplemented with 4.5 gm/I glucose, 20 mm glutamine, 20% fetal calfserum and with an anti-foaming agent, such aspolyoxyethylene-polyoxypropylene block copolymer.

In addition, antibody-producing cell lines can also be created bytechniques other than fusion, such as direct transformation of Blymphocytes with oncogenic DNA, or transfection with an oncovirus, suchas Epstein-Barr virus (EBV, also called human herpesvirus 4 (HHV-4)) orKaposi's sarcoma-associated herpesvirus (KSHV). See, U.S. Pat. Nos.4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917;4,472,500; 4,491,632; 4,493,890. A monoclonal antibody may also beproduced via an anti-receptor peptide or peptides containing thecarboxyl terminal as described well-known in the art. See Niman et al.,Proc. Natl. Acad. Sci. USA, 80: 4949-4953 (1983); Geysen et al., Proc.Natl. Acad. Sci. USA, 82: 178-182 (1985); Lei et al. Biochemistry34(20): 6675-6688, (1995). Typically, the anti-receptor peptide or apeptide analog is used either alone or conjugated to an immunogeniccarrier, as the immunogen for producing anti-receptor peptide monoclonalantibodies.

There are also a number of other well-known techniques for makingmonoclonal antibodies as binding molecules in this invention.Particularly useful are methods of making fully human antibodies. Onemethod is phage display technology which can be used to select a rangeof human antibodies binding specifically to the antigen using methods ofaffinity enrichment. Phage display has been thoroughly described in theliterature and the construction and screening of phage display librariesare well known in the art, see, e.g., Dente et al, Gene. 148(1):7-13(1994); Little et al, Biotechnol Adv. 12(3):539-55 (1994); Clackson etal., Nature 352:264-628 (1991); Huse et al., Science 246:1275-1281(1989).

Monoclonal antibodies derived by hybridoma technique from anotherspecies than human, such as mouse, can be humanized to avoid humananti-mouse antibodies when infused into humans. Among the more commonmethods of humanization of antibodies are complementarity-determiningregion grafting and resurfacing. These methods have been extensivelydescribed, see e.g. U.S. Pat. Nos. 5,859,205 and 6,797,492; Liu et al,Immunol Rev. 222:9-27 (2008); Almagro et 35 al, Front Biosci. 1;13:1619-33 (2008); Lazar et al, Mol Immunol. 44(8):1986-98 (2007); Li etal, Proc. Natl. Acad. Sci. USA. 103(10):3557-62 (2006) each incorporatedherein by reference. Fully human antibodies can also be prepared byimmunizing transgenic mice, rabbits, monkeys, or other mammals, carryinglarge portions of the human immunoglobulin heavy and light chains, withan immunogen. Examples of such mice are: the Xenomouse. (Abgenix, Inc.),the HuMAb-Mouse (Medarex/BMS), the VelociMouse (Regeneron), see alsoU.S. Pat. Nos. 6,596,541, 6,207,418, 6,150,584, 6,111,166, 6,075,181,5,922,545, 5,661,016, 5,545,806, 5,436,149 and 5,569,825. In humantherapy, murine variable regions and human constant regions can also befused to construct called “chimeric antibodies” that are considerablyless immunogenic in man than murine mAbs (Kipriyanov et al, MolBiotechnol. 26:39-60 (2004); Houdebine, Curr Opin Biotechnol. 13:625-9(2002) each incorporated herein by reference). In addition,site-directed mutagenesis in the variable region of an antibody canresult in an antibody with higher affinity and specificity for itsantigen (Brannigan et al, Nat Rev Mol Cell Biol. 3:964-70, (2002));Adams et al, J Immunol Methods. 231:249-60 (1999)) and exchangingconstant regions of a mAb can improve its ability to mediate effectorfunctions of binding and cytotoxicity.

Antibodies immunospecific for a malignant cell antigen can also beobtained commercially or produced by any method known to one of skill inthe art such as, e.g., chemical synthesis or recombinant expressiontechniques. The nucleotide sequence encoding antibodies immunospecificfor a malignant cell antigen can be obtained commercially, e.g., fromthe GenBank database or a database like it, the literature publications,or by routine cloning and sequencing.

Apart from an antibody, a peptide or protein that bind/block/target orin some other way interact with the epitopes or corresponding receptorson a targeted cell can be used as a binding molecule. These peptides orproteins could be any random peptide or proteins that have an affinityfor the epitopes or corresponding receptors and they don't necessarilyhave to be of the immunoglobulin family. These peptides can be isolatedby similar techniques as for phage display antibodies (Szardenings, JRecept Signal Transduct Res. 2003; 23(4):307-49). The use of peptidesfrom such random peptide libraries can be similar to antibodies andantibody fragments. The binding molecules of peptides or proteins may beconjugated on or linked to a large molecules or materials, such as, butis not limited, an albumin, a polymer, a liposome, a nano particle,dendrimers, as long as such attachment permits the peptide or protein toretain its antigen binding specificity.

Examples of antibodies used for conjugation of antimitotic agents inthis prevention for treating cancer, autoimmune disease, and infectiousdisease include, but are not limited to, 3F8 (anti-OD2), Abagovomab(anti CA-125), Abciximab (anti CD41 (integrin alpha-IIb), Adalimumab(anti-TNF-α), Adecatumumab (anti-EpCAM, CD326), Afelimomab (anti-TNF-α);Afutuzumab (anti-CD20), Alacizumab pegol (anti-VEGFR2), ALD518(anti-IL-6), Alemtuzumab (Campath, MabCampath, anti-CD52), Altumomab(anti-CEA), Anatumomab (anti-TAG-72), Anrukinzumab (IMA-638,anti-IL-13), Apolizumab (anti-HLA-DR), Arcitumomab (anti-CEA),Aselizumab (anti-L-selectin (CD62L), Atlizumab (tocilizumab, Actemra,RoActemra, anti-IL-6 receptor), Atorolimumab (anti-Rhesus factor),Bapineuzumab (anti-beta amyloid), Basiliximab (Simulect, antiCD25 (achain of IL-2 receptor), Bavituximab (anti-phosphatidylserine),Bectumomab (LymphoScan, anti-CD22), Belimumab (Benlysta, LymphoStat-B,anti-BAFF), Benralizumab (anti-CD125), Bertilimumab (anti-CCL11(eotaxin-1)), Besilesomab (Scintimun, anti-CEA-related antigen),Bevacizumab (Avastin, anti-VEGF-A), Biciromab (FibriScint, anti-fibrinII beta chain), Bivatuzumab (anti-CD44 v6), Blinatumomab (BiTE,anti-CD19), Brentuximab (cAC1O, anti-CD30 TNFRSF8), Briakinumab(anti-IL-12, IL-23) Canakinumab (Ilaris, anti-IL-1), Cantuzumab (C242,anti-CanAg), Capromab, Catumaxomab (Removab, anti-EpCAM, anti-CD3), CC49(anti-TAG-72), Cedelizumab (anti-CD4), Certolizumab pegol (Cimziaanti-TNF-α), Cetuximab (Erbitux, IMC-C225, anti-EGFR), Citatuzumabbogatox (anti-EpCAM), Cixutumumab (anti-IGF-1), Clenoliximab (anti-CD4),Clivatuzumab (anti-MUC1), Conatumumab (anti-TRAIL-R2), CR6261(anti-Influenza A hemagglutinin), Dacetuzumab (anti-CD40), Daclizumab(Zenapax, anti-CD25 (a chain of IL-2 receptor)), Daratumumab (anti-CD38(cyclic ADP ribose hydrolase), Denosumab (Prolia, anti-RANKL), Detumomab(anti-B-lymphoma cell), Dorlimomab, Dorlixizumab, Ecromeximab (anti-OD3ganglioside), Eculizumab (Soliris, anti-CS), Edobacomab(anti-endotoxin), Edrecolomab (Panorex, Mab17-1A, anti-EpCAM),Efalizumab (Raptiva, anti-LFA-1 (CD11a), Efungumab (Mycograb,anti-Hsp90), Elotuzumab (anti-SLAMF7), Elsilimomab (anti-IL-6),Enlimomab pegol (anti-ICAM-1 (CD54)), Epitumomab (anti-episialin),Epratuzumab (anti-CD22), Erlizumab (anti-ITGB2 (CD18)), Ertumaxomab(Rexomun, anti-HER2/neu, CD3), Etaracizumab (Abegrin, anti-integrinα_(v)β₃), Exbivirumab (anti-hepatitis B surface antigen), Fanolesomab(NeutroSpec, anti-CD15), Faralimomab (anti-interferon receptor),Farletuzumab (anti-folate receptor 1), Felvizumab (anti-respiratorysyncytial virus), Fezakinumab (anti-IL-22), Figitumumab (anti-IGF-1receptor), Fontolizumab (anti-IFN-γ), Foravirumab (anti-rabies virusglycoprotein), Fresolimumab (anti-TGF-β), Galiximab (anti-CD80),Gantenerumab (anti-beta amyloid), Gavilimomab (anti-CD147 (basigin)),Gemtuzumab (anti-CD33), Girentuximab (anti-carbonic anhydrase 9),Glembatumumab (CR011, anti-GPNMB), Golimumab (Simponi, anti-TNF-α),Gomiliximab (anti-CD23 (IgE receptor)), Ibalizumab (anti-CD4),Ibritumomab (anti-CD20), Igovomab (Indimacis-125, anti-CA-125),Imciromab (Myoscint, anti-cardiac myosin), Infliximab (Remicade,anti-TNF-α), Intetumumab (anti-CD51), Inolimomab (anti-CD25 (a chain ofIL-2 receptor)), Inotuzumab (anti-CD22), Ipilimumab (anti-CD152),Iratumumab (anti-CD30 (TNFRSF8)), Keliximab (anti-CD4), Labetuzumab(CEA-Cide, anti-CEA), Lebrikizumab (anti-IL-13), Lemalesomab(anti-NCA-90 (granulocyte antigen)), Lerdelimumab (anti-TGF beta 2),Lexatumumab (anti-TRAIL-R2), Libivirumab (anti-hepatitis B surfaceantigen), Lintuzumab (anti-CD33), Lucatumumab (anti-CD40), Lumiliximab(anti-CD23 (IgE receptor), Mapatumumab (anti-TRAIL-R1), Maslimomab(anti-T-cell receptor), Matuzumab (anti-EGFR), Mepolizumab (Bosatria,anti-IL-5), Metelimumab (anti-TGF beta 1), Milatuzumab (anti-CD74),Minretumomab (anti-TAG-72), Mitumomab (BEC-2, anti-OD3 ganglioside),Morolimumab (anti-Rhesus factor), Motavizumab (Numax, anti-respiratorysyncytial virus), Muromonab-CD3 (Orthoclone OKT3, anti-CD3), Nacolomab(anti-C242), Naptumomab (anti-5T4), Natalizumab (Tysabri, anti-integrinα₄), Nebacumab (anti-endotoxin), Necitumumab (anti-EGFR), Nerelimomab(anti-TNF-α), Nimotuzumab (Theracim, Theraloc, anti-EGFR), Nofetumomab,Ocrelizumab (anti-CD20), Odulimomab (Afolimomab, anti-LFA-1 (CD11a)),Ofatumumab (Arzerra, anti-CD20), Olaratumab (anti-PDGF-R α), Omalizumab(Xolair, anti-IgE Fe region), Oportuzumab (anti-EpCAM), Oregovomab(OvaRex, anti-CA-125), Otelixizumab (anti-CD3), Pagibaximab(anti-lipoteichoic acid), Palivizumab (Synagis, Abbosynagis,anti-respiratory syncytial virus), Panitumumab (Vectibix, ABX-EGF,anti-EGFR), Panobacumab (anti-Pseudomonas aeruginosa), Pascolizumab(anti-IL-4), Pemtumomab (Theragyn, anti-MUC1), Pertuzumab (Omnitarg,2C4, anti-HER2/neu), Pexelizumab (anti-CS), Pintumomab(anti-adenocarcinoma antigen), Priliximab (anti-CD4), Pritumumab(anti-vimentin), PRO 140 (anti-CCR5), Racotumomab (1E10,anti-(N-glycolylneuraminic acid (NeuGc, NGNA)-gangliosides GM3)),Rafivirumab (anti-rabies virus glycoprotein), Ramucirumab (anti-VEGFR2),Ranibizumab (Lucentis, anti-VEGF-A), Raxibacumab (anti-anthrax toxin,protective antigen), Regavirumab (anti-cytomegalovirus glycoprotein B),Reslizumab (anti-IL-5), Rilotumumab (anti-HOF), Rituximab (MabThera,Rituxanmab, anti-CD20), Robatumumab (anti-IGF-1 receptor), Rontalizumab(anti-IFN-α), Rovelizumab (LeukArrest, anti-CD11, CD18), RuplizumabAntova, anti-CD154 (CD40L)), Satumomab (anti-TAG-72), Sevirumab(anti-cytomegalovirus), Sibrotuzumab (anti-FAP), Sifalimumab(anti-IFN-α), Siltuximab (anti-IL-6), Siplizumab (anti-CO2), (Smart)MI95 (anti-CD33), Solanezumab (anti-beta amyloid), Sonepcizumab(anti-sphingosine-1-phosphate), Sontuzumab (anti-episialin), Stamulumab(anti-myostatin), Sulesomab (LeukoScan, (anti-NCA-90 (granulocyteantigen), Tacatuzumab (anti alpha-fetoprotein), Tadocizumab(anti-integrin α_(IIb)β₃), Talizumab (anti-IgE), Tanezumab (anti-NGF),Taplitumomab (anti-CD19), Tefibazumab (Aurexis, (anti-clumping factorA), Telimomab, Tenatumomab (anti-tenascin C), Teneliximab (anti-CD40),Teplizumab (anti-CD3), TGN1412 (anti-CD28), Ticilimumab (Tremelimumab,(anti-CTLA-4), Tigatuzumab (anti-TRAIL-R2), TNX-650 (anti-IL-13),Tocilizumab (Atlizumab, Actemra, RoActemra, (anti-IL-6 receptor),Toralizumab (anti-CD154 (CD40L)), Tositumomab (anti-CD20), Trastuzumab(Herceptin, (anti-HER2/neu), Tremelimumab (anti-CTLA-4), Tucotuzumabcelmoleukin (anti-EpCAM), Tuvirumab (anti-hepatitis B virus),Urtoxazumab (anti-Escherichia coli), Ustekinumab (Stelara, anti-IL-12,IL-23), Vapaliximab (anti-AOC3 (VAP-1)), Vedolizumab, (anti-integrinα₄β₇), Veltuzumab (anti-CD20), Vepalimomab (anti-AOC3 (VAP-1),Visilizumab (Nuvion, anti-CD3), Vitaxin (anti-vascular integrin avb3),Volociximab (anti-integrin α₅β₁), Votumumab (HumaSPECT, anti-tumorantigen CTAA16.88), Zalutumumab (HuMax-EGFr, (anti-EGFR), Zanolimumab(HuMax-CD4, anti-CD4), Ziralimumab (anti-CD147 (basigin)), Zolimomab(anti-CD5), Etanercept (Enbrel®), Alefacept (Amevive®), Abatacept(Orencia®), Rilonacept (Arcalyst), 14F7 [anti-IRP-2 (Iron RegulatoryProtein 2)], 14G2a (anti-GD2 ganglioside, from Nat. Cancer Inst. formelanoma and solid tumors), J591 (anti-PSMA, Weill Cornell MedicalSchool for prostate cancers), 225.28S [anti-HMW-MAA (High molecularweight-melanoma-associated antigen), Sorin Radiofarmaci S.R.L. (Milan,Italy) for melanoma], COL-1 (anti-CEACAM3, CGM1, from Nat. Cancer Inst.USA for colorectal and gastric cancers), CYT-356 (Oncoltad®, forprostate cancers), HNK20 (Ora Vax Inc. for respiratory syncytial virus),ImmuRAIT (from Immunomedics for NHL), Lym-1 (anti-HLA-DR10, PeregrinePharm. for Cancers), MAK-195F [anti-TNF (tumor necrosis factor; TNFA,TNF-alpha; TNFSF2), from Abbott/Knoll for Sepsis toxic shock], MEDI-500[T10B9, anti-CD3, TRaP3 (T cell receptor alpha/beta), complex, fromMedimmune Inc for Graft-versus-host disease], RING SCAN [anti-TAG 72(tumour associated glycoprotein 72), from Neoprobe Corp. for Breast,Colon and Rectal cancers], Avicidin (anti-EPCAM (epithelial celladhesion molecule), anti-TACSTD1 (Tumor-associated calcium signaltransducer 1), anti-GA733-2 (gastrointestinal tumor-associated protein2), anti-EGP-2 (epithelial glycoprotein 2); anti-KSA; KS1/4 antigen;M4S; tumor antigen 17-1A; CD326, from NeoRx Corp. for Colon, Ovarian,Prostate cancers and NHL]; LymphoCide (Immunomedics, NJ), Smart ID10(Protein Design Labs), Oncolym (Techniclone Inc, CA), Allomune(BioTransplant, CA), anti-VEGF (Genentech, CA); CEAcide (Immunomedics,NJ), IMC-1C11 (ImClone Systems, NJ) and Cetuximab (ImClone, NJ).

Other antibodies as binding ligands include, but are not limited to, areantibodies against the following antigens: Aminopeptidase N (CD13),Annexin A1, B7-H3 (CD276, various cancers), CA125, CA15-3 (carcinomas),CA19-9 (carcinomas), L6 (carcinomas), Lewis Y (carcinomas), Lewis X(carcinomas), alpha fetoprotein (carcinomas), CA242, placental alkalinephosphatase (carcinomas), prostate specific antigen (prostate),prostatic acid phosphatase (prostate), epidermal growth factor(carcinomas), CD2 (Hodgkin's disease, NHL lymphoma, multiple myeloma),CD3 epsilon (T cell lymphoma, lung, breast, gastric, ovarian cancers,autoimmune diseases, malignant ascites), CD19 (B cell malignancies),CD20 (non-Hodgkin's lymphoma), CD22 (leukemia, lymphoma, multiplemyeloma, SLE), CD30, CD33, CD38 (multiple myeloma), CD40 (lymphoma,multiple myeloma, leukemia), CD51 (Metastatic melanoma, sarcoma), CD52,CD56 (small cell lung cancers, ovarian cancer, Merkel cell carcinoma,and the liquid tumor, multiple myeloma), CD66e (cancers), CD70(metastatic renal cell carcinoma and non-Hodgkin lymphoma), CD74(multiple myeloma), CD80 (lymphoma), CD98 (cancers), mucin (carcinomas),CD221 (solid tumors), CD227 (breast, ovarian cancers), CD262 (NSCLC andother cancers), CD309 (ovarian cancers), CD326 (solid tumors), CEACAM3(colorectal, gastric cancers), CEACAM5 (carcinoembryonic antigen; CEA,CD66e) (breast, colorectal and lung cancers), DLL4 (A-like-4), EGFR(Epidermal Growth Factor Receptor, various cancers), CTLA4 (melanoma),CXCR4 (CD184, Heme-oncology, solid tumors), Endoglin (CD105, solidtumors), EPCAM (epithelial cell adhesion molecule, bladder, head, neck,colon, NHL prostate, and ovarian cancers), ERBB2 (Epidermal GrowthFactor Receptor 2; lung, breast, prostate cancers), FCGR1 (autoimmunediseases), FOLR (folate receptor, ovarian cancers), GD2 ganglioside(cancers), G-28 (a cell surface antigen glyvolipid, melanoma), GD3idiotype (cancers), Heat shock proteins (cancers), HER1 (lung, stomachcancers), HER2 (breast, lung and ovarian cancers), HLA-DR10 (NHL),HLA-DRB (NHL, B cell leukemia), human chorionic gonadotropin(carcinoma), IGF1R (insulin-like growth factor I receptor, solid tumors,blood cancers), IL-2 receptor (interleukin 2 receptor, T-cell leukemiaand lymphomas), IL-6R (interleukin 6 receptor, multiple myeloma, RA,Castleman's disease, IL6 dependent tumors), Integrins (αvβ3, α5β1, α6β4,α11β3, α5β5, αvβ5, for various cancers), MAGE-I (carcinomas), MAGE-2(carcinomas), MAGE-3 (carcinomas), MAGE 4 (carcinomas), anti-transferrinreceptor (carcinomas), p97 (melanoma), MS4A1 (membrane-spanning4-domains subfamily A member 1, Non-Hodgkin's B cell lymphoma,leukemia), MUC1 or MUC1-KLH (breast, ovarian, cervix, bronchus andgastrointestinal cancer), MUC16 (CA125) (Ovarian cancers), CEA(colorectal), gp100 (melanoma), MARTI (melanoma), MPG (melanoma), MS4A1(membrane-spanning 4-domains subfamily A, small cell lung cancers, NHL),Nucleolin, Neu oncogene product (carcinomas), P21 (carcinomas), Paratopeof anti-(N-glycolylneuraminic acid, Breast, Melanoma cancers), PLAP-liketesticular alkaline phosphatase (ovarian, testicular cancers), PSMA(prostate tumors), PSA (prostate), ROBO4, TAG 72 (tumour associatedglycoprotein 72, AML, gastric, colorectal, ovarian cancers), T celltransmembrane protein (cancers), Tie (CD202b), TNFRSF10B (tumor necrosisfactor receptor superfamily member 10B, cancers), TNFRSF13B (tumornecrosis factor receptor superfamily member 13B, multiple myeloma, NHL,other cancers, RA and SLE), TPBG (trophoblast glycoprotein, Renal cellcarcinoma), TRAIL-R¹ (Tumor necrosis apoprosis Inducing ligand Receptor1, lymphoma, NHL, colorectal, lung cancers), VCAM-1 (CD106, Melanoma),VEGF, VEGF-A, VEGF-2 (CD309) (various cancers). Some other tumorassociated antigens recognized by antibodies have been reviewed (Gerber,et al, mAbs 1:3, 247-253 (2009); Novellino et al, Cancer ImmunolImmunother. 54(3), 187-207 (2005). Franke, et al, Cancer BiotherRadiopharm. 2000, 15, 459-76). Many other antigens are: many otherCluster of Differentiations (CD4, CDS, CD6, CD7, CD8, CD9, CD10, CD11a,CD11 b, CD11c, CD12w, CD14, CD15, CD16, CDwl7, CD18, CD21, CD23, CD24,CD25, CD26, CD27, CD28, CD29, CD31, CD32, CD34, CD35, CD36, CD37, CD41,CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49b, CD49c, CD53, CD54,CD55, CD58, CD59, CD61, CD62E, CD62L, CD62P, CD63, CD68, CD69, CD71,CD72, CD79, CD79a, CD79b, CD81, CD82, CD83, CD86, CD87, CD88, CD89,CD90, CD91, CD95, CD96, CD100, CD103, CD105, CD106, CD109, CD117, CD120,CD127, CD133, CD134, CD135, CD138, CD141, CD142, CD143, CD144, CD147,CD151, CD152, CD154, CD156, CD158, CD163, CD166, CD168, CD184, CDw186,CD195, CD202 (a, b), CD209, CD235a, CD271, CD303, CD304), Apo2, ASLG659,BMPR1B (bone morphogenetic protein receptor), CRIPTO, Annexin A1,Nucleolin, Endoglin (CD105), ROBO4, Amino-peptidase N, Δ-like-4 (DLL4),VEGFR-2 (CD309), CXCR4 9CD184), Tie2, B7-H3, WT1, MUC1, LMP2, HPV E6 E7,EGFRvIII, HER-2/neu, Idiotype, MAGE A3, p53 nonmutant, NY-ESO-1, GD2,CEA, MelanA/MART1, Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrierfamily 34, member 2, type II sodium-dependent phosphate transporter 3b),Ras mutant, gp100, p53 mutant, Proteinase3 (PR¹), bcr-abl,Tetratocarcinoma-derived growth factors), EphA receptors, EphBreceptors, EGFr, EGFRvIII, ETBR (Endothelin), HER2/neu, HER3, HLA-DOB(MHC class II molecule Ia antigen), integrins, IRTA2, MPF (MPF, MSLN,SMR, megakaryocyte potentiating factor, mesothelin), cripto, Sema Sb(FLJ10372, KIAA1445, Mm42015, SEMA5B, 5EMAG, semaphoring 5 bHlog, sdemadomain, seven thrombospondin repeats, cytoplasmic domain), PSCA, STEAP1(six transmembrane epithelial antigen of prostate), and STEAP2 (HGNC8639, IPCA-1, PCANP1, STAMP1, STEAP2, STMP, prostate)Tyrosinase,Survivin, hTERT, Sarcoma translocation breakpoints, EphA2, PAP, ML-IAP,AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, Androgenreceptor, Cyclin B1, Polysialic acid, MYCN, RhoC, TRP-2, GD3, FucosylGM1, Mesothelin, PSCA, MAGE A1, sLe(a), CYP1B1, PLAC1, GM3, BORIS, Tn,GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX,PAX5, OY-TES1, Sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1,B7H3, Legumain, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-13, MAD-CT-2,Fas-related antigen 1.

In another specific embodiment, the antimitotic agent-binding moleculeconjugates of the invention are used in accordance with the compositionsand methods of the invention for the treatment of cancers. The cancersinclude, but are not limited, Adrenocortical Carcinoma, Anal Cancer,Bladder Cancer, Brain Tumor (Adult, Brain Stem Glioma, Childhood,Cerebellar Astrocytoma, Cerebral Astrocytoma, Ependymoma,Medulloblastoma, Supratentorial Primitive Neuroectodermal and PinealTumors, Visual Pathway and Hypothalamic Glioma), Breast Cancer,Carcinoid Tumor, Gastrointestinal, Carcinoma of Unknown Primary,Cervical Cancer, Colon Cancer, Endometrial Cancer, Esophageal Cancer,Extrahepatic Bile Duct Cancer, Ewings Family of Tumors (PNET),Extracranial Germ Cell Tumor, Eye Cancer, Intraocular Melanoma,Gallbladder Cancer, Gastric Cancer (Stomach), Germ Cell Tumor,Extragonadal, Gestational Trophoblastic Tumor, Head and Neck Cancer,Hypopharyngeal Cancer, Islet Cell Carcinoma, Kidney Cancer (renal cellcancer), Laryngeal Cancer, Leukemia (Acute Lymphoblastic, Acute Myeloid,Chronic Lymphocytic, Chronic Myelogenous, Hairy Cell), Lip and OralCavity Cancer, Liver Cancer, Lung Cancer (Non-Small Cell, Small Cell,Lymphoma (AIDS-Related, Central Nervous System, Cutaneous T-Cell,Hodgkin's Disease, Non-Hodgkin's Disease, Malignant Mesothelioma,Melanoma, Merkel Cell Carcinoma, Metasatic Squamous Neck Cancer withOccult Primary, Multiple Myeloma, and Other Plasma Cell Neoplasms,Mycosis Fungoides, Myelodysplastic Syndrome, MyeloproliferativeDisorders, Nasopharyngeal Cancer, Neuroblastoma, Oral Cancer,Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer (Epithelial, GermCell Tumor, Low Malignant Potential Tumor), Pancreatic Cancer (Exocrine,Islet Cell Carcinoma), Paranasal Sinus and Nasal Cavity Cancer,Parathyroid Cancer, Penile Cancer, Pheochromocytoma Cancer, PituitaryCancer, Plasma Cell Neoplasm, Prostate Cancer Rhabdomyosarcoma, RectalCancer, Renal Cell Cancer (kidney cancer), Renal Pelvis and Ureter(Transitional Cell), Salivary Gland Cancer, Sezary Syndrome, SkinCancer, Skin Cancer (Cutaneous T-Cell Lymphoma, Kaposi's Sarcoma,Melanoma), Small Intestine Cancer, Soft Tissue Sarcoma, Stomach Cancer,Testicular Cancer, Thymoma (Malignant), Thyroid Cancer, Urethral Cancer,Uterine Cancer (Sarcoma), Unusual Cancer of Childhood, Vaginal Cancer,Vulvar Cancer, Wilms' Tumor

In another specific embodiment, the antimitotic agent-binding moleculeconjugates of the invention are used in accordance with the compositionsand methods of the invention for the treatment or prevention of anautoimmune disease. The autoimmune diseases include, but are notlimited, Achlorhydra Autoimmune Active Chronic Hepatitis, AcuteDisseminated Encephalomyelitis, Acute hemorrhagic leukoencephalitis,Addison's Disease, Agammaglobulinemia, Alopecia areata, AmyotrophicLateral Sclerosis, Ankylosing Spondylitis, Anti-GBMITBM Nephritis,Antiphospholipid syndrome, Antisynthetase syndrome, Arthritis, Atopicallergy, Atopic Dermatitis, Autoimmune Aplastic Anemia, Autoimmunecardiomyopathy, Autoimmune hemolytic anemia, Autoimmune hepatitis,Autoimmune inner ear disease, Autoimmune lymphoproliferative syndrome,Autoimmune peripheral neuropathy, Autoimmune pancreatitis, Autoimmunepolyendocrine syndrome Types I, II, & III, Autoimmune progesteronedermatitis, Autoimmune thrombocytopenic purpura, Autoimmune uveitis,Balo disease/Balo concentric sclerosis, Bechets Syndrome, Berger'sdisease, Bickerstaffs encephalitis, Blau syndrome, Bullous Pemphigoid,Castleman's disease, Chagas disease, Chronic Fatigue Immune DysfunctionSyndrome, Chronic inflammatory demyelinating polyneuropathy, Chronicrecurrent multifocal ostomyelitis, Chronic lyme disease, Chronicobstructive pulmonary disease, Churg-Strauss syndrome, CicatricialPemphigoid, Coeliac Disease, Cogan syndrome, Cold agglutinin disease,Complement component 2 deficiency, Cranial arteritis, CREST syndrome,Crohns Disease (a type of idiopathic inflammatory bowel diseases),Cushing's Syndrome, Cutaneous leukocytoclastic angiitis, Dego's disease,Dercum's disease, Dermatitis herpetiformis, Dermatomyositis, Diabetesmellitus type 1, Diffuse cutaneous systemic sclerosis, Dressler'ssyndrome, Discoid lupus erythematosus, Eczema, Endometriosis,Enthesitis-related arthritis, Eosinophilic fasciitis, Epidermolysisbullosa acquisita, Erythema nodosum, Essential mixed cryoglobulinemia,Evan's syndrome, Fibrodysplasia ossificans progressiva, Fibromyalgia,Fibromyositis, Fibrosing aveolitis, Gastritis, Gastrointestinalpemphigoid, Giant cell arteritis, Glomerulonephritis, Goodpasture'ssyndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto'sencephalitis, Hashimoto's thyroiditis, Haemolytic anaemia,Henoch-Schonlein purpura, Herpes gestationis, Hidradenitis suppurativa,Hughes syndrome (See Antiphospholipid syndrome), Hypogammaglobulinemia,Idiopathic Inflammatory Demyelinating Diseases, Idiopathic pulmonaryfibrosis, Idiopathic thrombocytopenic purpura (See Autoimmunethrombocytopenic purpura), IgA nephropathy (Also Berger's disease),Inclusion body myositis, Inflammatory demyelinating polyneuopathy,Interstitial cystitis, Irritable Bowel Syndrome, Juvenile idiopathicarthritis, Juvenile rheumatoid arthritis, Kawasaki's Disease,Lambert-Eaton myasthenic syndrome, Leukocytoclastic vasculitis, Lichenplanus, Lichen sclerosus, Linear IgA disease (LAD), Lou Gehrig's Disease(Also Amyotrophic lateral sclerosis), Lupoid hepatitis, Lupuserythematosus, Majeed syndrome, Meniere's disease, Microscopicpolyangiitis, Miller-Fisher syndrome, Mixed Connective Tissue Disease,Morphea, Mucha-Habermann disease, Muckle-Wells syndrome, MultipleMyeloma, Multiple Sclerosis, Myasthenia gravis, Myositis, Narcolepsy,Neuromyelitis optica (Devic's Disease), Neuromyotonia, Occularcicatricial pemphigoid, Opsoclonus myoclonus syndrome, Ord thyroiditis,Palindromic rheumatism, PANDAS (Pediatric Autoimmune NeuropsychiatricDisorders Associated with Streptococcus), Paraneoplastic cerebellardegeneration, Paroxysmal nocturnal hemoglobinuria, Parry Rombergsyndrome, Parsonnage-Turner syndrome, Pars planitis, Pemphigus,Pemphigus vulgaris, Pernicious anaemia, Perivenous encephalomyelitis,POEMS syndrome, Polyarteritis nodosa, Polymyalgia rheumatica,Polymyositis, Primary biliary cirrhosis, Primary sclerosing cholangitis,Progressive inflammatory neuropathy, Psoriasis, Psoriatic Arthritis,Pyoderma gangrenosum, Pure red cell aplasia, Rasmussen's encephalitis,Raynaud phenomenon, Relapsing polychondritis, Reiter's syndrome,Restless leg syndrome, Retroperitoneal fibrosis, Rheumatoid arthritis,Rheumatoid fever, Sarcoidosis, Schizophrenia, Schmidt syndrome,Schnitzler syndrome, Scleritis, Scleroderma, Sjögren's syndrome,Spondyloarthropathy, Sticky blood syndrome, Still's Disease, Stiffperson syndrome, Subacute bacterial endocarditis, Susac's syndrome,Sweet syndrome, Sydenham Chorea, Sympathetic ophthalmia, Takayasu'sarteritis, Temporal arteritis (giant cell arteritis), Tolosa-Huntsyndrome, Transverse Myelitis, Ulcerative Colitis (a type of idiopathicinflammatory bowel diseases), Undifferentiated connective tissuedisease, Undifferentiated spondyloarthropathy, Vasculitis, Vitiligo,Wegener's granulomatosis, Wilson's syndrome, Wiskott-Aldrich syndrome

In another specific embodiment, a binding molecule used for theconjugate for the treatment or prevention of an autoimmune diseaseincludes, but are not limited to, anti-elastin antibody; Abys againstepithelial cells antibody; Anti-Basement Membrane Collagen Type IVProtein antibody; Anti-Nuclear Antibody; Anti ds DNA; Anti ss DNA, AntiCardiolipin Antibody IgM, IgG; anti-celiac antibody; Anti PhospholipidAntibody IgK, IgG; Anti SM Antibody; Anti Mitochondrial Antibody;Thyroid Antibody; Microsomal Antibody, T-cells antibody; ThyroglobulinAntibody, Anti SCL-70; Anti-Jo; Anti-U.sub.1RNP; Anti-La/SSB; Anti SSA;Anti SSB; Anti Perital Cells Antibody; Anti Histones; Anti RNP; C-ANCA;P-ANCA; Anti centromere; Anti-Fibrillarin, and Anti GBM Antibody,Anti-ganglioside antibody; Anti-Desmogein 3 antibody; Anti-p62 antibody;Anti-sp100 antibody; Anti-Mitochondrial (M2) antibody; Rheumatoid factorantibody; Anti-MCV antibody; Anti-topoisomerase antibody;Anti-neutrophil cytoplasmic (cANCA) antibody.

In certain preferred embodiments, the binding molecule for the conjugatein the present invention, can bind to either a receptor or a receptorcomplex expressed on an activated lymphocyte which is associated with anautoimmune disease. The receptor or receptor complex can comprise animmunoglobulin gene superfamily member (e.g. CD2, CD3, CD4, CD8, CD19,CD22, CD28, CD79, CD90, CD152/CTLA-4, PD-1, or ICOS), a TNF receptorsuperfamily member (e.g. CD27, CD40, CD95/Fas, CD134/OX40, CD137/4-1BB,INF-R1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2/TRAIL-R¹,TRAIL-R², TRAIL-R³, TRAIL-R⁴, and APO-3), an integrin, a cytokinereceptor, a chemokine receptor, a major histocompatibility protein, alectin (C-type, S-type, or I-type), or a complement control protein.

In another specific embodiment, useful binding ligands that areimmunospecific for a viral or a microbial antigen are humanized or humanmonoclonal antibodies. As used herein, the term “viral antigen”includes, but is not limited to, any viral peptide, polypeptide protein(e.g. HIV gp120, HIV nef, RSV F glycoprotein, influenza virusneuraminidase, influenza virus hemagglutinin, HTLV tax, herpes simplexvirus glycoprotein (e.g. gB, gC, gD, and gE) and hepatitis B surfaceantigen) that is capable of eliciting an immune response. As usedherein, the term “microbial antigen” includes, but is not limited to,any microbial peptide, polypeptide, protein, saccharide, polysaccharide,or lipid molecule (e.g., a bacteria, fungi, pathogenic protozoa, oryeast polypeptide including, e.g., LPS and capsular polysaccharide 5/8)that is capable of eliciting an immune response. Examples of antibodiesavailable 1 for the viral or microbial infection include, but are notlimited to, Palivizumab which is a humanized anti-respiratory syncytialvirus monoclonal antibody for the treatment of RSV infection; PRO542which is a CD4 fusion antibody for the treatment of HIV infection;Ostavir which is a human antibody for the treatment of hepatitis Bvirus; PROTVIR which is a humanized IgG. sub. 1 antibody for thetreatment of cytomegalovirus; and anti-LPS antibodies.

The binding molecules-antimitotic agent conjugates of this invention canbe used in the treatment of infectious diseases. These infectiousdiseases include, but are not limited to, Acinetobacter infections,Actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS(Acquired immune deficiency syndrome), Amebiasis, Anaplasmosis, Anthrax,Arcano-bacterium haemolyticum infection, Argentine hemorrhagic fever,Ascariasis, Aspergillosis, Astrovirus infection, Babesiosis, Bacilluscereus infection, Bacterial pneumonia, Bacterial vaginosis, Bacteroidesinfection, Balantidiasis, Baylisascaris infection, BK virus infection,Black piedra, Blastocystis hominis infection, Blastomycosis, Bolivianhemorrhagic fever, Borrelia infection, Botulism (and Infant botulism),Brazilian hemorrhagic fever, Brucellosis, Burk-holderia infection,Buruli ulcer, Calicivirus infection (Norovirus and Sapovirus),Campylo-bacteriosis, Candidiasis (Moniliasis; Thrush), Cat-scratchdisease, Cellulitis, Chagas Disease (American trypanosomiasis),Chancroid, Chickenpox, Chlamydia, Chlamydophila pneumoniae infection,Cholera, Chromoblastomycosis, Clonorchiasis, Clostridium difficileinfection, Coccidioidomycosis, Colorado tick fever, Common cold (Acuteviral rhinopharyngitis; Acute coryza), Creutzfeldt-Jakob disease,Crimean-Congo hemorrhagic fever, Cryptococcosis, Cryptosporidiosis,Cutaneous larva migrans, Cyclosporiasis, Cysticercosis, Cytomegalovirusinfection, Dengue fever, Dientamoebiasis, Diphtheria,Diphyllobothriasis, Dracunculiasis, Ebola hemorrhagic fever,Echinococcosis, Ehrlichiosis, Enterobiasis (Pinworm infection),Enterococcus infection, Enterovirus infection, Epidemic typhus, Erythemainfectiosum (Fifth disease), Exanthem subitum, Fasciolopsiasis,Fasciolosis, Fatal familial insomnia, Filariasis, Food poisoning byClostridium perfringens, Free-living amebic infection, Fusobacteriuminfection, Gas gangrene (Clostridial myonecrosis), Geotrichosis,Gerstmann-Straussler-Scheinker syndrome, Giardiasis, Glanders,Gnathostomiasis, Gonorrhea, Granuloma inguinale (Donovanosis), Group Astreptococcal infection, Group B streptococcal infection, Haemophilusinfluenzae infection, Hand, foot and mouth disease (HFMD), HantavirusPulmonary Syndrome, Helicobacter pylori infection, Hemolytic-uremicsyndrome, Hemorrhagic fever with renal syndrome, Hepatitis A, HepatitisB, Hepatitis C, Hepatitis D, Hepatitis E, Herpes simplex,Histoplasmosis, Hookworm infection, Human bocavirus infection, Humanewingii ehrlichiosis, Human granulocytic anaplasmosis, Humanmetapneumovirus infection, Human monocytic ehrlichiosis, Humanpapillomavirus infection, Human parainfluenza virus infection,Hymenolepiasis, Epstein-Barr Virus Infectious Mononucleosis (Mono),Influenza, Isosporiasis, Kawasaki disease, Keratitis, Kingella kingaeinfection, Kuru, Lassa fever, Legionellosis (Legionnaires' disease),Legionellosis (Pontiac fever), Leishmaniasis, Leprosy, Leptospirosis,Listeriosis, Lyme disease (Lyme borreliosis), Lymphatic filariasis(Elephantiasis), Lymphocytic choriomeningitis, Malaria, Marburghemorrhagic fever, Measles, Melioidosis (Whitmore's disease),Meningitis, Meningococcal disease, Metagonimiasis, Microsporidiosis,Molluscum contagiosum, Mumps, Murine typhus (Endemic typhus), Mycoplasmapneumonia, Mycetoma, Myiasis, Neonatal conjunctivitis (Ophthalmianeonatorum), (New) Variant Creutzfeldt-Jakob disease (vCJD, nvCJD),Nocardiosis, Onchocerciasis (River blindness), Paracoccidioidomycosis(South American blastomycosis), Paragonimiasis, Pasteurellosis,Pediculosis capitis (Head lice), Pediculosis corporis (Body lice),Pediculosis pubis (Pubic lice, Crab lice), Pelvic inflammatory disease,Pertussis (Whooping cough), Plague, Pneumococcal infection, Pneumocystispneumonia, Pneumonia, Poliomyelitis, Prevotella infection, Primaryamoebic meningoencephalitis, Progressive multifocal leukoencephalopathy,Psittacosis, Q fever, Rabies, Rat-bite fever, Respiratory syncytialvirus infection, Rhinosporidiosis, Rhinovirus infection, Rickettsialinfection, Rickettsialpox, Rift Valley fever, Rocky mountain spottedfever, Rotavirus infection, Rubella, Salmonellosis, SARS (Severe AcuteRespiratory Syndrome), Scabies, Schistosomiasis, Sepsis, Shigellosis(Bacillary dysentery), Shingles (Herpes zoster), Smallpox (Variola),Sporotrichosis, Staphylococcal food poisoning, Staphylococcal infection,Strongyloidiasis, Syphilis, Taeniasis, Tetanus (Lockjaw), Tinea barbae(Barber's itch), Tinea capitis (Ringworm of the Scalp), Tinea corporis(Ringworm of the Body), Tinea cruris (Jock itch), Tinea manuum, Tineanigra, Tinea pedis (Athlete's foot), Tinea unguium (Onychomy-cosis),Tinea versicolor (Pityriasis versicolor), Toxocariasis (Ocular LarvaMigrans), Toxocariasis (Visceral Larva Migrans), Toxoplasmosis,Trichinellosis, Trichomoniasis, Trichuriasis (Whipworm infection),Tuberculosis, Tularemia, Ureaplasma urealyticum infection, Venezuelanequine encephalitis, Venezuelan hemorrhagic fever, Viral pneumonia, WestNile Fever, White piedra (Tinea blanca), Yersinia pseudotuberculosisinfection, Yersiniosis, Yellow fever, Zygomycosis.

The binding molecules, preferable antibodies described in this patentthat are against pathogenic strains include, but are not limit,Acinetobacter baumannii, Actinomyces israelii, Actinomyces gerencseriaeand Propionibacterium propionicus, Trypanosoma brucei, HIV (Humanimmunodeficiency virus), Entamoeba histolytica, Anaplasma genus,Bacillus anthracis, Arcanobacterium haemolyticum, Junin virus, Ascarislumbricoides, Aspergillus genus, Astroviridae family, Babesia genus,Bacillus cereus, multiple bacteria, Bacteroides genus, Balantidium coli,Baylisascaris genus, BK virus, Piedraia hortae, Blastocystis hominis,Blastomyces dermatitides, Machupo virus, Borrelia genus, Clostridiumbotulinum, Sabia, Brucella genus, usually Burkholderia cepacia and otherBurkholderia species, Mycobacterium ulcerans, Caliciviridae family,Campylobacter genus, usually Candida albicans and other Candida species,Bartonella henselae, Group A Streptococcus and Staphylococcus,Trypanosoma cruzi, Haemophilus ducreyi, Varicella zoster virus (VZV),Chlamydia trachomatis, Chlamydophila pneumoniae, Vibrio cholerae,Fonsecaea pedrosoi, Clonorchis sinensis, Clostridium difficile,Coccidioides immitis and Coccidioides posadasii, Colorado tick fevervirus, rhinoviruses, coronaviruses, CJD prion, Crimean-Congo hemorrhagicfever virus, Cryptococcus neoformans, Cryptosporidium genus, Ancylostomabraziliense; multiple parasites, Cyclospora cayetanensis, Taenia solium,Cytomegalovirus, Dengue viruses (DEN-1, DEN-2, DEN-3 andDEN-4)—Flaviviruses, Dientamoeba fragilis, Corynebacterium diphtheriae,Diphyllobothrium, Dracunculus medinensis, Ebolavirus, Echinococcusgenus, Ehrlichia genus, Enterobius vermicularis, Enterococcus genus,Enterovirus genus, Rickettsia prowazekii, Parvovirus B19, Humanherpesvirus 6 and Human herpesvirus 7, Fasciolopsis buski, Fasciolahepatica and Fasciola gigantica, FFI prion, Filarioidea superfamily,Clostridium perfringens, Fusobacterium genus, Clostridium perfringens;other Clostridium species, Geotrichum candidum, GSS prion, Giardiaintestinalis, Burkholderia mallei, Gnathostoma spinigerum andGnathostoma hispidum, Neisseria gonorrhoeae, Klebsiella granulomatis,Streptococcus pyogenes, Streptococcus agalactiae, Haemophilusinfluenzae, Enteroviruses, mainly Coxsackie A virus and Enterovirus 71,Sin Nombre virus, Helicobacter pylori, Escherichia coli O157:H7,Bunyaviridae family, Hepatitis A Virus, Hepatitis B Virus, Hepatitis CVirus, Hepatitis D Virus, Hepatitis E Virus, Herpes simplex virus 1,Herpes simplex virus 2, Histoplasma capsulatum, Ancylostoma duodenaleand Necator americanus, Hemophilus influenzae, Human bocavirus,Ehrlichia ewingii, Anaplasma phagocytophilum, Human metapneumovirus,Ehrlichia chaffeensis, Human papillomavirus, Human parainfluenzaviruses, Hymenolepis nana and Hymenolepis diminuta, Epstein-Barr Virus,Orthomyxoviridae family, Isospora belli, Kingella kingae, Klebsiellapneumoniae, Klebsiella ozaenas, Klebsiella rhinoscleromotis, Kuru prion,Lassa virus, Legionella pneumophila, Legionella pneumophila, Leishmaniagenus, Mycobacterium leprae and Mycobacterium lepromatosis, Leptospiragenus, Listeria monocytogenes, Borrelia burgdorferi and other Borreliaspecies, Wuchereria bancrofti and Brugia malayi, Lymphocyticchoriomeningitis virus (LCMV), Plasmodium genus, Marburg virus, Measlesvirus, Burkholderia pseudomallei, Neisseria meningitides, Metagonimusyokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV),Mumps virus, Rickettsia typhi, Mycoplasma pneumoniae, numerous speciesof bacteria (Actinomycetoma) and fungi (Eumycetoma), parasitic dipterousfly larvae, Chlamydia trachomatis and Neisseria gonorrhoeae, vCJD prion,Nocardia asteroides and other Nocardia species, Onchocerca volvulus,Paracoccidioides brasiliensis, Paragonimus westermani and otherParagonimus species, Pasteurella genus, Pediculus humanus capitis,Pediculus humanus corporis, Phthirus pubis, Bordetella pertussis,Yersinia pestis, Streptococcus pneumoniae, Pneumocystis jirovecii,Poliovirus, Prevotella genus, Naegleria fowleri, JC virus, Chlamydophilapsittaci, Coxiella burnetii, Rabies virus, Streptobacillus moniliformisand Spirillum minus, Respiratory syncytial virus, Rhinosporidiumseeberi, Rhinovirus, Rickettsia genus, Rickettsia akari, Rift Valleyfever virus, Rickettsia rickettsii, Rotavirus, Rubella virus, Salmonellagenus, SARS coronavirus, Sarcoptes scabiei, Schistosoma genus, Shigellagenus, Varicella zoster virus, Variola major or Variola minor,Sporothrix schenckii, Staphylococcus genus, Staphylococcus genus,Staphylococcus aureus, Streptococcus pyogenes, Strongyloidesstercoralis, Treponema pallidum, Taenia genus, Clostridium tetani,Trichophyton genus, Trichophyton tonsurans, Trichophyton genus,Epidermophyton floccosum, Trichophyton rubrum, and Trichophytonmentagrophytes, Trichophyton rubrum, Hortaea werneckii, Trichophytongenus, Malassezia genus, Toxocara canis or Toxocara cati, Toxoplasmagondii, Trichinella spiralis, Trichomonas vaginalis, Trichuristrichiura, Mycobacterium tuberculosis, Francisella tularensis,Ureaplasma urealyticum, Venezuelan equine encephalitis virus, Vibriocolerae, Guanarito virus, West Nile virus, Trichosporon beigelii,Yersinia pseudotuberculosis, Yersinia enterocolitica, Yellow fevervirus, Mucorales order (Mucormycosis) and Entomophthorales order(Entomophthoramycosis), Pseudomonas aeruginosa, Campylobacter (Vibrio)fetus, Aeromonas hydrophila, Edwardsiella tarda, Yersinia pestis,Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonellatyphimurium, Treponema pertenue, Treponema carateneum, Borreliavincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae,Pneumocystis carinii, Brucella abortus, Brucella suis, Brucellamelitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsiatsutsugumushi, Clamydia spp.; pathogenic fungi (Aspergillus fumigatus,Candida albicans, Histoplasma capsulatum); protozoa (Entomoebahistolytica, Trichomonas tenas, Trichomonas hominis, Tryoanosomagambiense, Trypanosoma rhodesiense, Leishmania donovani, Leishmaniatropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodiumvivax, Plasmodium falciparum, Plasmodium malaria); or Helminiths(Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium,and hookworms).

Other antibodies as a binding ligand in this invention for treatment ofviral disease include, but are not limited to, antibodies againstantigens of pathogenic viruses, including as examples and not bylimitation: Poxyiridae, Herpesviridae, Adenoviridae, Papovaviridae,Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae, Retroviridae,influenza viruses, parainfluenza viruses, mumps, measles, respiratorysyncytial virus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae,Non-A/Non-B Hepatitis virus, Rhinoviridae, Coronaviridae, Rotoviridae,Oncovirus [such as, HBV (Hepatocellular carcinoma), HPV (Cervicalcancer, Anal cancer), Kaposi's sarcoma-associated herpesvirus (Kaposi'ssarcoma), Epstein-Barr virus (Nasopharyngeal carcinoma, Burkitt'slymphoma, Primary central nervous system lymphoma), MCPyV (Merkel cellcancer), SV40 (Simian virus 40), HCV (Hepatocellular carcinoma), HTLV-I(Adult T-cell leukemia/lymphoma)], Immune disorders caused virus: [suchas Human Immunodeficiency Virus (AIDS)]; Central nervous system virus:[such as, JCV (Progressive multifocal leukoencephalopathy), MeV(Subacute sclerosing panencephalitis), LCV (Lymphocyticchoriomeningitis), Arbovirus encephalitis, Orthomyxoviridae (probable)(Encephalitis lethargica), RV (Rabies), Chandipura virus, Herpesviralmeningitis, Ramsay Hunt syndrome type II; Poliovirus (Poliomyelitis,Post-polio syndrome), HTLV-I (Tropical spastic paraparesis)];Cytomegalovirus (Cytomegalovirus retinitis, HSV (Herpetic keratitis));Cardiovascular virus [such as CBV (Pericarditis, Myocarditis)];Respiratory system/acute viral nasopharyngitis/viral pneumonia:[Epstein-Barr virus (EBV infection/Infectious mononucleosis),Cytomegalovirus; SARS coronavirus (Severe acute respiratory syndrome)Orthomyxoviridae: Influenza virus A/B/C (Influenza/Avian influenza),Paramyxovirus: Human parainfluenza viruses (Parainfluenza), RSV (Humanrespiratory syncytial virus), hMPV]; Digestive system virus [MuV(Mumps), Cytomegalovirus (Cytomegalovirus esophagitis); Adenovirus(Adenovirus infection); Rotavirus, Norovirus, Astrovirus, Coronavirus;HBV (Hepatitis B virus), CBV, HAV (Hepatitis A virus), HCV (Hepatitis Cvirus), HDV (Hepatitis D virus), HEV (Hepatitis E virus), HGV (HepatitisG virus)]; Urogenital virus [such as, BK virus, MuV (Mumps)].

According to a further object, the present invention also concernspharmaceutical compositions comprising the conjugate of the inventiontogether with a pharmaceutically acceptable carrier for treatment ofcancer and autoimmune disorders. The method for treatment of cancer andautoimmune disorders can be practiced in vitro, in vivo, or ex vivo.Examples of in vitro uses include treatments of cell cultures in orderto kill all cells except for desired variants that do not express thetarget antigen; or to kill variants that express undesired antigen.Examples of ex vivo uses include treatments of hematopoietic stem cells(HSC) prior to the performance of the transplantation (HSCT) into thesame patient in order to kill diseased or malignant cells. For instance,clinical ex vivo treatment to remove tumour cells or lymphoid cells frombone marrow prior to autologous transplantation in cancer treatment orin treatment of autoimmune disease, or to remove T cells and otherlymphoid cells from allogeneic bone marrow or tissue prior to transplantin order to prevent graft-versus-host disease, can be carried out asfollows. Bone marrow is harvested from the patient or other individualand then incubated in medium containing serum to which is added theconjugate of the invention, concentrations range from about 1 pM to 0.1mM, for about 30 minutes to about 48 hours at about 37° C. The exactconditions of concentration and time of incubation (=dose) are readilydetermined by the skilled clinicians. After incubation the bone marrowcells are washed with medium containing serum and returned to thepatient by i.v. infusion according to known methods. In circumstanceswhere the patient receives other treatment such as a course of ablativechemotherapy or total-body irradiation between the time of harvest ofthe marrow and reinfusion of the treated cells, the treated marrow cellsare stored frozen in liquid nitrogen using standard medical equipment.

For clinical in vivo use, the conjugate of the invention will besupplied as solutions or as a lyophilized solid that can be redisolvedin sterile water for injection. Examples of suitable protocols ofconjugate administration are as follows. Conjugates are given weekly for8 weeks as an i.v. bolus. Bolus doses are given in 50 to 500 ml ofnormal saline to which human serum albumin (e.g. 0.5 to 1 mL of aconcentrated solution of human serum albumin, 100 mg/mL) can be added.Dosages will be about 50 μg to 20 mg/kg of body weight per week, i.v.(range of 10 μg to 200 mg/kg per injection). 8 weeks after treatment,the patient may receive a second course of treatment. Specific clinicalprotocols with regard to route of administration, excipients, diluents,dosages, times, etc., can be determined by the skilled clinicians.

Examples of medical conditions that can be treated according to the invivo or ex vivo methods of killing selected cell populations includemalignancy of any types of cancer, autoimmune diseases, graftrejections, and infections (viral, bacterial or parasite).

The amount of a conjugate which is required to achieve the desiredbiological effect, will vary depending upon a number of factors,including the chemical characteristics, the potency, and thebioavailability of the conjugates, the type of disease, the species towhich the patient belongs, the diseased state of the patient, the routeof administration, all factors which dictate the required dose amounts,delivery and regimen to be administered.

In general terms, the conjugates of this invention may be provided in anaqueous physiological buffer solution containing 0.1 to 10% w/vconjugates for parenteral administration. Typical dose ranges are from 1μg/kg to 0.1 g/kg of body weight per day; a preferred dose range is from0.01 mg/kg to 20 mg/kg of body weight per day or an equivalent dose in ahuman child. The preferred dosage of drug to be administered is likelyto depend on such variables as the type and extent of progression of thedisease or disorder, the overall health status of the particularpatient, the relative biological efficacy of the compound selected, theformulation of the compound, the route of administration (intravenous,intramuscular, or other), the pharmaco-kinetic properties of thecompound by the chosen delivery route, and the speed (bolus orcontinuous infusion) and schedule of administrations (number ofrepetitions in a given period of time).

The conjugates of the present invention are also capable of beingadministered in unit dose forms, wherein the term “unit dose” means asingle dose which is capable of being administered to a patient, andwhich can be readily handled and packaged, remaining as a physically andchemically stable unit dose comprising either the active conjugateitself, or as a pharmaceutically acceptable composition, as describedhereinafter. As such, typical total daily dose ranges are from 0.01 to100 mg/kg of body weight. By way of general guidance, unit doses forhumans range from 1 mg to 3000 mg per day. Preferably the unit doserange is from 1 to 500 mg administered one to four times a day, and evenmore preferably from 10 mg to 500 mg, once a day. Conjugates providedherein can be formulated into pharmaceutical compositions by admixturewith one or more pharmaceutically acceptable excipients. Such unit dosecompositions may be prepared for use by oral administration,particularly in the form of tablets, simple capsules or soft gelcapsules; or intranasally, particularly in the form of powders, nasaldrops, or aerosols; or dermally, for example, topically in ointments,creams, lotions, gels or sprays, or via trans-dermal patches. Thecompositions may conveniently be administered in unit dosage form andmay be prepared by any of the methods well known in the pharmaceuticalart, for example, as described in Remington: The Science and Practice ofPharmacy, 21^(th) ed.; Lippincott Williams & Wilkins: Philadelphia, Pa.,2005.

Preferred formulations include pharmaceutical compositions in which acompound of the present invention is formulated for oral or parenteraladministration. For oral administration, tablets, pills, powders,capsules, troches and the like can contain one or more of any of thefollowing ingredients, or compounds of a similar nature: a binder suchas microcrystalline cellulose, or gum tragacanth; a diluent such asstarch or lactose; a disintegrant such as starch and cellulosederivatives; a lubricant such as magnesium stearate; a glidant such ascolloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin; or a flavoring agent such as peppermint, or methylsalicylate. Capsules can be in the form of a hard capsule or softcapsule, which are generally made from gelatin blends optionally blendedwith plasticizers, as well as a starch capsule. In addition, dosage unitforms can contain various other materials that modify the physical formof the dosage unit, for example, coatings of sugar, shellac, or entericagents. Other oral dosage forms syrup or elixir may contain sweeteningagents, preservatives, dyes, colorings, and flavorings. In addition, theactive compounds may be incorporated into fast dissolve,modified-release or sustained-release preparations and formulations, andwherein such sustained-release formulations are preferably bi-modal.Preferred tablets contain lactose, cornstarch, magnesium silicate,croscarmellose sodium, povidone, magnesium stearate, or talc in anycombination.

Liquid preparations for parenteral administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions. The liquidcompositions may also include binders, buffers, preservatives, chelatingagents, sweetening, flavoring and coloring agents, and the like.Non-aqueous solvents include alcohols, propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and organic esters such asethyl oleate. Aqueous carriers include mixtures of alcohols and water,buffered media, and saline. In particular, biocompatible, biodegradablelactide polymer, lactide/glycolide copolymer, orpolyoxyethylene-polyoxypropylene copolymers may be useful excipients tocontrol the release of the active compounds. Intravenous vehicles caninclude fluid and nutrient replenishers, electrolyte replenishers, suchas those based on Ringer's dextrose, and the like. Other potentiallyuseful parenteral delivery systems for these active compounds includeethylene-vinyl acetate copolymer particles, osmotic pumps, implantableinfusion systems, and liposomes.

Alternative modes of administration include formulations for inhalation,which include such means as dry powder, aerosol, or drops. They may beaqueous solutions containing, for example, polyoxyethylene-9-laurylether, glycocholate and deoxycholate, or oily solutions foradministration in the form of nasal drops, or as a gel to be appliedintranasally. Formulations for buccal administration include, forexample, lozenges or pastilles and may also include a flavored base,such as sucrose or acacia, and other excipients such as glycocholate.Formulations suitable for rectal administration are preferably presentedas unit-dose suppositories, with a solid based carrier, such as cocoabutter, and may include a salicylate. Formulations for topicalapplication to the skin preferably take the form of an ointment, cream,lotion, paste, gel, spray, aerosol, or oil. Carriers which can be usedinclude petroleum jelly, lanolin, polyethylene glycols, alcohols, ortheir combinations. Formulations suitable for transdermal administrationcan be presented as discrete patches and can be lipophilic emulsions orbuffered, aqueous solutions, dissolved and/or dispersed in a polymer oran adhesive.

In a specific embodiment, a conjugate of the invention is administeredconcurrently with the other known or will be known therapeutic agentssuch as the chemotherapeutic agent, the radiation therapy, immunotherapyagents, autoimmune disorder agents, anti-infectious agents or the otherantibody-drug conjugates, resulting in a synergistic effect. In anotherspecific embodiment, the synergistic drugs or radiation therapy areadministered prior or subsequent to administration of a conjugate, inone aspect at least an hour, 12 hours, a day, a week, a month, infurther aspects several months, prior or subsequent to administration ofa conjugate of the invention.

In other embodiments, the synergistic drugs include, but not limited to:

1). Chemotherapeutic agents: a). Alkylating agents: such as [Nitrogenmustards: (chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine,melphalan, trofosfamide); Nitrosoureas: (carmustine, lomustine);Alkylsulphonates: (busulfan, treosulfan); Triazenes: (dacarbazine);Platinum containing compounds: (carboplatin, cisplatin, oxaliplatin)];b). Plant Alkaloids: such as [Vinca alkaloids: (vincristine,vinblastine, vindesine, vinorelbine); Taxoids: (paclitaxel, docetaxol)];c). DNA Topoisomerase Inhibitors: such as [Epipodophyllins:(9-aminocamptothecin, camptothecin, crisnatol, etoposide, etoposidephosphate, irinotecan, teniposide, topotecan); Mitomycins: (mitomycinC)]; d). Anti-metabolites: such as {[Anti-folate: DHFR inhibitors:(methotrexate, trimetrexate); IMP dehydrogenase Inhibitors:(mycophenolic acid, tiazofurin, ribavirin, EICAR); Ribonucleotidereductase Inhibitors: (hydroxyurea, deferoxamine)]; [Pyrimidine analogs:Uracil analogs: (5-Fluorouracil, doxifluridine, floxuridine, ratitrexed(Tomudex)); Cytosine analogs: (cytarabine, cytosine arabinoside,fludarabine); Purine analogs: (azathioprine, mercaptopurine,thioguanine)]}; e). Hormonal therapies: such as {Receptor antagonists:[Anti-estrogen: (megestrol, raloxifene, tamoxifen); LHRH agonists:(goscrclin, leuprolide acetate); Anti-androgens: (bicalutamide,flutamide)]; Retinoids/Deltoids: [Vitamin D3 analogs: (CB 1093, EB 1089KH 1060, cholecalciferol, ergocalciferol); Photodynamic therapies:(verteporfin, phthalocyanine, photosensitizer Pc4, dimethoxy-hypocrellinA); Cytokines: (Interferon-alpha, Interferon-gamma, tumor necrosisfactor (TNFs), human proteins containing a TNF domain)]}; f). Kinaseinhibitors, such as BIBW 2992 (anti-EGFR/Erb2), imatinib, gefitinib,pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib,lapatinib, axitinib, pazopanib. vandetanib, E7080 (anti-VEGFR2),mubritinib, ponatinib (AP24534), bafetinib (INNO-406), bosutinib(SKI-606), cabozantinib, vismodegib, iniparib, ruxolitinib, CYT387,axitinib, tivozanib, sorafenib, bevacizumab, cetuximab, Trastuzumab,Ranibizumab, Panitumumab, ispinesib; g). Others: such as gemcitabine,epoxomicins (e.g. carfilzomib), bortezomib, thalidomide, lenalidomide,pomalidomide, tosedostat, zybrestat, PLX4032, STA-9090, Stimuvax,allovectin-7, Xegeva, Provenge, Yervoy, Isoprenylation inhibitors (suchas Lovastatin), Dopaminergic neurotoxins (such as1-methyl-4-phenylpyridinium ion), Cell cycle inhibitors (such asstaurosporine), Actinomycins (such as Actinomycin D, dactinomycin),Bleomycins (such as bleomycin A2, bleomycin B2, peplomycin),Anthracyclines (such as daunorubicin, doxorubicin (adriamycin),idarubicin, epirubicin, pirarubicin, zorubicin, mtoxantrone, MDRinhibitors (such as verapamil), Ca²⁺ ATPase inhibitors (such asthapsigargin), Histone deacetylase inhibitors (Vorinostat, Romidepsin,Panobinostat, Valproic acid, Mocetinostat (MGCD0103), Belinostat,PCI-24781, Entinostat, SB939, Resminostat, Givinostat, AR-42, CUDC-101,sulforaphane, Trichostatin A); Thapsigargin, Celecoxib, glitazones,epigallocatechin gallate, Disulfiram, Salinosporamide A. 2). Ananti-autoimmune disease agent includes, but is not limited to,cyclosporine, cyclosporine A, aminocaproic acid, azathioprine,bromocriptine, chlorambucil, chloroquine, cyclophosphamide,corticosteroids (e.g. amcinonide, betamethasone, budesonide,hydrocortisone, flunisolide, fluticasone propionate, fluocortolonedanazol, dexamethasone, Triamcinolone acetonide, beclometasonedipropionate), DHEA, enanercept, hydroxyl-chloroquine, infliximab,meloxicam, methotrexate, mofetil, mycophenylate, prednisone, sirolimus,tacrolimus.

3). An anti-infectious disease agent includes, but is not limited to,a). Aminoglycosides: amikacin, astromicin, gentamicin (netilmicin,sisomicin, isepamicin), hygromycin B, kanamycin (amikacin, arbekacin,bekanamycin, dibekacin, tobramycin), neomycin (framycetin, paromomycin,ribostamycin), netilmicin, spectinomycin, streptomycin, tobramycin,verdamicin; b). Amphenicols: azidamfenicol, chloramphenicol,florfenicol, thiamphenicol; c). Ansamycins: geldanamycin, herbimycin;d). Carbapenems: biapenem, doripenem, ertapenem, imipenem/cilastatin,meropenem, panipenem; e). Cephems: carbacephem (loracarbef),cefacetrile, cefaclor, cefradine, cefadroxil, cefalonium, cefaloridine,cefalotin or cefalothin, cefalexin, cefaloglycin, cefamandole,cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin,cefbuperazone, cefcapene, cefdaloxime, cefepime, cefminox, cefoxitin,cefprozil, cefroxadine, ceftezole, cefuroxime, cefixime, cefdinir,cefditoren, cefepime, cefetamet, cefmenoxime, cefodizime, cefonicid,cefoperazone, ceforanide, cefotaxime, cefotiam, cefozopran, cephalexin,cefpimizole, cefpiramide, cefpirome, cefpodoxime, cefprozil, cefquinome,cefsulodin, ceftazidime, cefteram, ceftibuten, ceftiolene, ceftizoxime,ceftobiprole, ceftriaxone, cefuroxime, cefuzonam, cephamycin (cefoxitin,cefotetan, cefmetazole), oxacephem (flomoxef, latamoxef); f).Glycopeptides: bleomycin, vancomycin (oritavancin, telavancin),teicoplanin (dalbavancin), ramoplanin, cubicin; g). Glycylcyclines: e.g.tigecycline; g). 13-Lactamase inhibitors: penam (sulbactam, tazobactam),clavam (clavulanic acid); i). Lincosamides: clindamycin, lincomycin; j).Lipopeptides: daptomycin, A54145, calcium-dependent antibiotics (CDA);k). Macrolides: azithromycin, cethromycin, clarithromycin,dirithromycin, erythromycin, flurithromycin, josamycin, ketolide(telithromycin, cethromycin), midecamycin, miocamycin, oleandomycin,rifamycins (rifampicin, rifampin, rifabutin, rifapentine), rokitamycin,roxithromycin, spectinomycin, spiramycin, tacrolimus (FK506),troleandomycin, telithromycin; 1). Monobactams: aztreonam, tigemonam;m). Oxazolidinones: linezolid; n). Penicillins: amoxicillin, ampicillin(pivampicillin, hetacillin, bacampicillin, metampicillin,talampicillin), azidocillin, azlocillin, benzylpenicillin, benzathinebenzylpenicillin, benzathine phenoxymethyl-penicillin, clometocillin,procaine benzylpenicillin, carbenicillin (carindacillin), cloxacillin,dicloxacillin, epicillin, flucloxacillin, mecillinam (pivmecillinam),mezlocillin, meticillin, nafcillin, oxacillin, penamecillin, penicillin,pheneticillin, phenoxymethylpenicillin, piperacillin, propicillin,sulbenicillin, temocillin, ticarcillin; o). Polypeptides: bacitracin,colistin, polymyxin B; p). Quinolones: alatrofloxacin, balofloxacin,ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin,enrofloxacin, floxin, garenoxacin, gatifloxacin, gemifloxacin,grepafloxacin, kano trovafloxacin, levofloxacin, lomefloxacin,marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin,ofloxacin, pefloxacin, trovafloxacin, grepafloxacin, sitafloxacin,sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin; q).Streptogramins: pristinamycin, quinupristin/dalfopristin); r).Sulfonamides: mafenide, prontosil, sulfacetamide, sulfamethizole,sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim,trimethoprim-sulfamethoxazole (co-trimoxazole); s). Steroidantibacterials: e.g. fusidic acid; t). Tetracyclines: doxycycline,chlortetracycline, clomocycline, demeclocycline, lymecycline,meclocycline, metacycline, minocycline, oxytetracycline,penimepicycline, rolitetracycline, tetracycline, glycylcyclines (e.g.tigecycline); u). Other types of antibiotics: annonacin, arsphenamine,bactoprenol inhibitors (Bacitracin), DADAL/AR inhibitors (cycloserine),dictyostatin, discodermolide, eleutherobin, epothilone, ethambutol,etoposide, faropenem, fusidic acid, furazolidone, isoniazid,laulimalide, metronidazole, mupirocin, mycolactone, NAM synthesisinhibitors (e.g. fosfomycin), nitrofurantoin, paclitaxel, platensimycin,pyrazinamide, quinupristin/dalfopristin, rifampicin (rifampin),tazobactam tinidazole, uvaricin;

4). Anti-viral drugs: a). Entry/fusion inhibitors: aplaviroc, maraviroc,vicriviroc, gp41 (enfuvirtide), PRO 140, CD4 (ibalizumab); b). Integraseinhibitors: raltegravir, elvitegravir, globoidnan A; c). Maturationinhibitors: bevirimat, vivecon; d). Neuraminidase inhibitors:oseltamivir, zanamivir, peramivir; e). Nucleosides &_nucleotides:abacavir, aciclovir, adefovir, amdoxovir, apricitabine, brivudine,cidofovir, clevudine, dexelvucitabine, didanosine (ddl), elvucitabine,emtricitabine (FTC), entecavir, famciclovir, fluorouracil (5-FU),3′-fluoro-substituted 2′,3′-dideoxynucleoside analogues (e.g.3′-fluoro-2′,3′-dideoxythymidine (FLT) and3′-fluoro-2′,3′-dideoxyguanosine (FLO), fomivirsen, ganciclovir,idoxuridine, lamivudine (3TC), 1-nucleosides (e.g. β-1-thymidine andβ-1-2′-deoxycytidine), penciclovir, racivir, ribavirin, stampidine,stavudine (d4T), taribavirin (viramidine), telbivudine, tenofovir,trifluridine valaciclovir, valganciclovir, zalcitabine (ddC), zidovudine(AZT); f). Non-nucleosides: amantadine, ateviridine, capravirine,diarylpyrimidines (etravirine, rilpivirine), delavirdine, docosanol,emivirine, efavirenz, foscarnet (phosphonoformic acid), imiquimod,interferon alfa, loviride, lodenosine, methisazone, nevirapine, NOV-205,peginterferon alfa, podophyllotoxin, rifampicin, rimantadine, resiquimod(R-848), tromantadine; g). Protease inhibitors: amprenavir, atazanavir,boceprevir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir,pleconaril, ritonavir, saquinavir, telaprevir (VX-950), tipranavir; h).Other types of anti-virus drugs: abzyme, arbidol, calanolide a,ceragenin, cyanovirin-n, diarylpyrimidines, epigallocatechin gallate(EGCG), foscarnet, griffithsin, taribavirin (viramidine), hydroxyurea,KP-1461, miltefosine, pleconaril, portmanteau inhibitors, ribavirin,seliciclib.

5). Other immunotheraphy drugs: e.g. imiquimod, interferons (e.g. α, β),granulocyte colony-stimulating factors, cytokines, Interleukins(IL-1-IL-35), antibodies (e.g. trastuzumab, pertuzumab, bevacizumab,cetuximab, panitumumab, infliximab, adalimumab, basiliximab, daclizumab,omalizumab), Protein-bound drugs (e.g., Abraxane), an antibodyconjugated with drugs selected from calicheamicin derivative, ofmaytansine derivatives (DM1 and DM4), CC-1065 and duocarmycin minorgroove binders, potent taxol derivatives, doxorubicin, auristatinantimitotic drugs (e.g. Trastuzumab-DM1, Inotuzumab ozogamicin,Brentuximab vedotin, Glembatumumab vedotin, lorvotuzumab mertansine,AN-152 LMB2, TP-38, VB4-845, Cantuzumab mertansine, AVE9633, SAR3419,CAT-8015 (anti-CD22), IMGN388, IMGN529, IMGN853,milatuzumab-doxorubicin, SGN-75 (anti-CD70), Anti-CD22-MCC-DM1).

According to a still further object, the present invention is alsoconcerned with the process of preparation of the conjugate of theinvention. The conjugate and process of the present invention may beprepared in a number of ways well known to those skilled in the art. Theantimitotic agents used in the conjugate can be synthesized, forexample, by application or adaptation of the methods described below, orvariations thereon as appreciated by the skilled artisan. Theappropriate modifications and substitutions will be readily apparent andwell known or readily obtainable from the scientific literature to thoseskilled in the art. In particular, such methods can be found in R. C.Larock, Comprehensive Organic Transformations, 2^(nd) Edition, Wiley-VCRPublishers, 1999.

In the reactions described hereinafter, it may be necessary to protectreactive functional groups, for example hydroxy, amino, imino, thio orcarboxy groups, where these are desired in the final product, to avoidtheir unwanted participation in the reactions. Conventional protectinggroups may be used in accordance with standard practice, for examplessee P. G. Wuts and T. W. Greene, Greene's Protective Groups in OrganicSynthesis, Wiley-Interscience; 4th edition (2006). Some reactions may becarried out in the presence of a base, or an acid or in a suitablesolvent. There is no particular restriction on the nature of the base,acid and solvent to be used in this reaction, and any base, acid orsolvent conventionally used in reactions of this type may equally beused here, provided that it has no adverse effect on other parts of themolecule. The reactions can take place over a wide range oftemperatures. In general, we find it convenient to carry out thereaction at a temperature of from −80° C. to 150° C. (more preferablyfrom about room temperature to 100° C.). The time required for thereaction may also vary widely, depending on many factors, notably thereaction temperature and the nature of the reagents. However, providedthat the reaction is effected under the preferred conditions outlinedabove, a period of from 3 hours to 20 hours will usually suffice.

The work-up of the reaction can be carried out by conventional means.For example, the reaction products may be recovered by distilling offthe solvent from the reaction mixture or, if necessary after distillingoff the solvent from the reaction mixture, pouring the residue intowater followed by extraction with a water-immiscible organic solvent anddistilling off the solvent from the extract. Additionally, the productcan, if desired, be further purified by various well known techniques,such as recrystallization, reprecipitation or the various chromatographytechniques, notably column chromatography or preparative thin layerchromatography. The synthesis of the antimitotic agents and theirconjugates of this invention are illustrated in the FIGS. 1-28.

The conjugates of binding molecules with potent antimitotic agents arefurther illustrated but not restricted by the description in thefollowing examples.

6. EXPERIMENTAL MATERIALS

Mass spectra were obtained using a Bruker Esquire 3000 system. NMRspectra were recorded on a Bruker AVANCE300 spectrometer. Chemicalshifts are reported in ppm relative to TMS as an internal standard.Ultraviolet spectra were recorded on a Hitachi U1200 spectrophotometer.HPLC was performed using an Agilent 1100 HPLC system equipped with afraction collector and a variable wavelength detector. Thin layerchromatography was performed on Analtech GF silica gel TLC plates.Aminal acids and their derivatives as well as preloaded resins wereeither from Merck Chemicals International Co, or Synthetech Co., orPeptides International Inc or Chembridge International Co. orSigma-Aldrich Co. Some of the linkers, Linkers of NHS ester/Maleimide(AMAS, BMPS, GMBS, MBS, SMCC, EMCS or Sulfo-EMCS, SMPB, SMPH, LC-SMCC,Sulfo-KMUS, SM(PEG)4, SM(PEG)6, SM(PEG)8, SM(PEG)12, SM(PEG)24); NHSester/Pyridyldithiol (SPDP, LC-SPDP or Sulfo-LC-SPDP, SMPT,Sulfo-LC-SMPT); NHS esters/Haloacetyl (SIA, SBAP, SIAB or Sulfo-SIAB);NHS ester/Diazirine (SDA or Sulfo-SDA, LC-SDA or Sulfo-LC-SDA, SDAD orSulfo-SDAD); Maleimide/Hydrazide (BMPH, EMCH, MPBH, KMUH);Pyridyldithiol/Hydrazide (PDPH); Isocyanate/Maleimide (PMPI) werepurchased from Thermo Fisher Scientific Co. SPDB, SPP linkers were madeaccording to the references (Cumber, A. et al, Bioconjugate Chem., 1992,3, 397-401). Human anti-CD22 antibody was from Santa Cruz Biotechnology,Inc. and Trastuzumab was from Genentech. All other chemicals oranhydrous solvents were from Sigma-Aldrich International.

Example 1. Methyl 4-(bis(2-hydroxyethyl)amino)-4-oxobutanoate (3)

Dimethyl succinate (20.0 g, 136.9 mmol) and dihydroxyethylamine (7.20 g,68.7 mmol) in the mixture of anhydrous toluene (500 ml) and pyridine (50ml) were refluxed at 150° C. for 28 h. The mixture was concentrated andpurified on SiO₂ column eluted with EtOAc/DCM (5% 25% EtOAc) to affordthe title compound (12.5 g, 83% yield). ESI MS m/z+ for C₉H₁₇NaNO₅(M+Na) cald 242.2, found 242.4.

Example 2. Methyl4-(bis(2-((methylsulfonyl)oxy)ethyl)amino)-4-oxobutanoate (4)

Methyl 4-(bis(2-hydroxyethyl)amino)-4-oxobutanoate (12.0 g, 49.56 mmol)in anhydrous pyridine (350 ml) was added methanesulfonyl chloride (20.0g, 175.4 mmol). After stirred overnight the mixture was concentrated,diluted with EtOAc (350 ml), washed with cold 1 M NaH₂PO₄ (2×300 ml),dried over MgSO₄, filtered and evaporated to afford crude product (˜18.8g, 101% yield). The crude product was used for next step without furtherpurification. ESI MS m/z+ for C₁₁H₂₁NaNO₉S₂(M+Na) cald 398.2, found398.4.

Example 3. Methyl 4-(bis(2-(acetylthio)ethyl)amino)-4-oxobutanoate (5)

Methyl 4-(bis(2-((methylsulfonyl)oxy)ethyl)amino)-4-oxobutanoate (freshmade, 90% pure, 8.5 g, ˜20 mmol) in DMA (350 ml) at 0° C. was addedthioacetic acid (10 ml, 134 mmol), followed by addtion of Et₃N (30 ml,215 mmol). The mixture was then stirred at room temperature overnight,concentrated, diluted with EtOAc (350 ml), washed with NaHCO₃ (sat, 300ml), NaCl sat solution (300 ml) and 1 M NaH₂PO₄ (300 ml). The organiclayer was dried over Na₂SO₄, filtered, evaporated and purified on SiO₂column eluted with EtOAc/hexane (10%-25% EtOAc) to afford the titlecompound (5.1 g, 76% yield). ESI MS m/z+ for C₁₃H₂₁NaNO₅S₂(M+Na) cald358.1, found 358.2.

Example 4. 4-(Bis(2-(pyridin-2-yldisulfanyl)ethyl)amino)-4-oxobutanoicacid (6)

Methyl 4-(bis(2-(acetylthio)ethyl)amino)-4-oxobutanoate (5.0 g, 14.9mmol) in THF (150 ml) was added NaOH (5.0 g, 125 mmol) in water (100ml). The mixture was stirred at RT for 35 min, neutralized with H₃PO₄ topH 7. Then 1,2-di(pyridin-2-yl)disulfane (Aldrithiol-2, 26.0 g, 118mmol) in THF (100 ml) was added and the mixture was stirred for 4 h,concentrated and purified on SiO₂ column eluted with MeOH/DCM/HOAc(1:20/1%) to afford the title product (5.8 g, 85.6% yield). ESI MS m/z+for C₁₈H₂₁NaN₃O₃S₄ (M+Na) cald 478.0, found 478.2.

Example 5. 2,5-dioxopyrrolidin-1-yl4-(bis(2-(pyridin-2-yldisulfanyl)ethyl)amino)-4-oxobutanoate (7)

4-(Bis(2-(pyridin-2-yldisulfanyl)ethyl)amino)-4-oxobutanoic acid (5.2 g,11.5 mmol) in DMA (100 ml) was added NHS (1.6 g, 13.9 mmol) and EDC (5.0g, 26.1 mmol). The mixture was stirred overnight, evaporated andpurified on SiO₂ column eluted with EtOAc/DCM (5% to 15% EtOAc) toafford the title product (5.8 g, 85.6% yield). ESI MS m/z+ forC₂₂H₂₄NaN₄O₅S₄ (M+Na) cald 575.1, found 575.2.

Example 6. 3,6-endoxo-Δ-tetrahydrophthalimide (12)

Maleimide (10.0 g, 103.0 mmol) in toluene (200 ml) was added furan (10.0ml, 137.4 mmol). The mixture was heated inside a 1 L of autoclave bombat 100° C. for 8 h. The bomb was cooled to room temperature, and theinside solid was rinsed with methanol, concentrated and crystallized inethyl acetate/hexane to afford 16.7 g (99%) of the title compound. 1HNMR (CDCl₃): 11.12 (s, 1H) (NH), 6.68-6.64 (m, 2H), 5.18-5.13 (m, 2H),2.97-2.92 (m, 2H). MS m/z+ for C₈H7NaNO₃ (M+Na) cald 188.04, found188.04.

Example 7. Methyl4-((2-((3aR,4R,7S,7aS)-1,3-dioxo-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindol-2(3H)-yl)ethyl)(2-((4R,7S,7aS)-1,3-dioxo-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindol-2(3H)-yl)ethyl)amino)-4-oxobutanoate(13)

Methyl 4-(bis(2-((methylsulfonyl)oxy)ethyl)amino)-4-oxobutanoate (4,fresh made, 90% pure, 8.5 g, −20 mmol) in DMA (350 ml) was added3,6-endoxo-Δ-tetrahydrophthalimide (10.2 g, 61.8 mmol), sodium carbonate(8.0 g, 75.5 mmol) and sodium iodide (0.3 g, 2.0 mmol). The mixture wasthen stirred at room temperature overnight, concentrated, diluted withEtOAc (350 ml), washed with NaHCO₃ (sat, 300 ml), NaCl sat solution (300ml) and 1 M NaH₂PO₄ (300 ml). The organic layer was dried over Na₂SO₄,filtered, evaporated and purified on SiO₂ column eluted withEtOAc/hexane (10%-30% EtOAc) to afford the title compound (7.9 g, 77%yield). ESI MS m/z+ for C₂₅H₂₇NaN₃O₉(M+Na) cald 536.2, found 536.4.

Example 8.4-(bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-4-oxobutanoicacid (14)

Compound 13 (3.0 g, 5.8 mmol) and trimethylstannanol (4.8 g, 26.4 mmol)in 1,2-dichloroethane (150 ml) was refluxed at 80° C. for 8 h. It wascooled to room temperature and the residue was passed a short silica gelcolumn and eluted with dichloromethane/methanol to remove the extratrimethyltin hydroxide. Then the pooled fractions were combined,concentrated and diluted with DMA and toluene, refluxed at 120° C.overnight and purified on SiO₂ column eluted with MeOH/DCM (5%-10% MeOH)to afford the title compound (1.62 g, 76% yield). ESI MS m/z+ forC₁₆H₁₇NaN₃O₉(M+Na) cald 386.1, found 386.2.

Example 9. 2,5-Dioxopyrrolidin-1-yl4-(bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-4-oxobutanoate

Compound (14)) (1.60 g, 4.4 mmol) in DMA (100 ml) was added NHS (0.76 g,6.61 mmol) and EDC (1.70 g, 8.90 mmol). The mixture was stirredovernight, evaporated and purified on SiO₂ column eluted with EtOAc/DCM(5% to 15% EtOAc) to afford the title product (1.72 g, 85.0% yield). ESIMS m/z+ for C₂₀H₂₀NaN₄O₉(M+Na) cald 483.1, found 483.2.

Example 10. t-Butyl 5-(3′,6′-endoxo-Δ-tetrahydrophthalimido) pentanoate

t-Butyl 5-hydroxy pentanote (10.0 g, 57.4 mmol) in pyridine (60 ml) wasadded mesyl chloride (8.0 ml, 103.3 mmol) and the mixture was stirredfor 6 h, evaporated, diluted with EtOAc, washed with cold 1M NaH₂PO₄, pH6, dried over MgSO₄, filtered and evaporated to dryness. To the mixtureof compound 12 (9.90 g, 60.0 mmol) and Na₂CO₃ (8.5 g, 80.1 mmol) in DMF(80 ml) was added the dryness mesylate compound. The mixture was stirredovernight, evaporated, diluted with EtOAc, washed with saline water and1M NaH₂PO₄, pH 6, dried over MgSO₄, evaporated and purified on SiO₂column eluted with EtOAc/CH₂Cl₂ (1:12) to afford the title compound(14.01 g, 76%). MS m/z+ for C₁₇H₂₃NaNO₅ (M+Na) cald 344.16, found344.16.

Example 11. 5-maleimido-pentanoic acid (21b)

Compound 17 (5.0 g, 15.57 mmol) in 1,4-dioxane (40 ml) was added HCl (10ml, 36%) at 4° C. and the mixture was stirred for 30 min, evaporated todryness to form 5-(3′,6′-endoxo-Δ-tetrahydrophthalimido) pentanoic acid(4.08 g, 99%). The dried compound in mixture of DMF/toluene (1:1, 40 ml)was refluxed for 6 h, evaporated and crystallized from EtOH/ether/hexaneto afford the title compound (2.76 g, 90%). MS m/z+ for C₉H₁₂NO₄ (M+H)cald 198.07, found 198.07.

Example 12. N-succinimidyl 5-maleimido-pentanoate (23b) (DMPS Linker)

5-maleimido-pentanoic acid 21b (2.0 g, 10.1 mmol) in CH₂Cl₂ (20 ml) wasadded N-hydroxysuccimide (1.50 g, 13.0 mmol) and EDC (7.0 g, 36.4 mmol)and the mixture was stirred overnight, evaporated and purified on SiO₂column (EtOAc/CH₂Cl₂, 1:10) to afford the title compound 23b (2.43 g,82%). MS m/z+ for C₁₃H₁₄NaN₂O₆(M+Na) cald 317.09, found 317.09.

Example 13. t-Butyl 5-(3′,6′-endoxo-Δ-tetrahydrophthalimido)pentanoylhydrazine-carboxylate (25a-a)

5-(3′,6′-endoxo-Δ-tetrahydrophthalimido) pentanoic acid (1.0 g, 3.77) inDMF (30 ml) was added tert-butyl carbazate (0.60 g, 4.53 mmol) and EDC(2.0 g, 10.4 mmol). The mixture was stirred overnight, evaporated andpurified on SiO₂ column (EtOAc/CH₂Cl₂, 1:10) to afford the titlecompound (1.18 g, 83%). MS m/z+ for C₁₈H₂₅NaN₃O₆(M+Na) cald 402.17,found 402.18.

Example 14. 5-maleimido-pentanoic acid hydrazide (25a-b)

Compound 25a-a (1.18 g, 3.11 mmol) was dissolved in the mixture ofDMF/toluene (1:1, 20 ml), refluxed for 6 h and envaporated. Then themixture dissolved in 1,4-dioxane (20 ml) was added HCl (5 ml, 36%) at 4°C. and the mixture was stirred for 30 min, evaporated to dryness andcrystallized from EtOH/ether/hexane to afford the title compound (577mg, 88%). MS m/z+ for C₉H₁₄N₃O₃ (M+H) cald 212.10, found 212.10.

Example 15. General Procedure for 3′-bromo-maleimdo Compounds 39 and 40,and 3′,4′-dibromo-maleimdo Compounds 43 and 44

Amino compound 37 or 38 (˜6 g) in DMF (60 ml) was added bromomaleicanhydride (1 eq) or 2,3-dibromomaleic anhydride (1 eq) and the mixturewas stirred overnight, evaporated via oil pump to dryness to afford thecrude enoic acids. To the crude enoic acids were added HOAc (˜50 ml) andAc2O (2-4 g) and the reaction mixture was fluxed at 120° C. for 6-12 h,concentrated and purified on SiO₂ column eluted with EtOAc/CH₂Cl₂(1:10-1:1) to afford (61%-87% yield) of the 3′-bromo-maleimdo compounds39 and 40, and 3′,4′-dibromomaleimdo compounds 43 and 44 respectively.

5-(3-bromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentanoic acid

MS m/z+ for C₉H₁₁BrNO₄ (M+H) cald 275.98, found 275.98.

3-(2-(2-(2-(3-bromo-2,5-dioxo-2,5-dihydro-1-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)-propanoicacid

MS m/z+ for C₁₃H₁₉BrNO₇ (M+H) cald 380.03, found 380.03.

5-(3,4-dibromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentanoic acid

MS m/z+ for C₉H₁₀Br₂NO₄ (M+H) cald 353.89, found 353.89.

3-(2-(2-(2-(3,4-dibromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)-propanoicacid

MS m/z+ for C₁₃H₁₈Br₂NO₇ (M+H) cald 457.94, found 457.94.

Example 16. General Procedure for NHS Ester of 3′-bromo-maleimdoCompounds 41 and 42, and NHS Ester of 3′,4′-dibromo-maleimdo Compounds45 and 46

R=(C₁˜C₈ alkyl or C₂H₄(OC₂H₄)_(n), n=1˜20; X³=H or Br 41, 42, X₃=H; 45,46, X₃=Br

To the solution of 3′-bromo-maleimdo compounds 39 and 40 (1 eq), or3′,4′-dibromo-maleimdo compounds 43 and 44 in DMA (˜0.15 M) were addedN-hydroxysuccinimide (1.1 eq) and EDC (2˜4 eq) and the mixture wasstirred overnight, concentrated and purified on SiO₂ column eluted withEtOAc/CH₂Cl₂ (1:20˜1:5) to afford (70%-93% yield) of the3′-bromo-maleimdo linkers 41 and 42, and 3′,4′-dibromo-maleimdo linkers45 and 46 respectively.

2,5-dioxopyrrolidin-1-yl5-(3-bromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentanoate

MS m/z+ for C₁₃H₁₃BrN₂NaO₇ (M+Na) cald 395.00, found 395.00.

2,5-dioxopyrrolidin-1-yl5-(3,4-dibromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentanoate

MS m/z+ for C₁₃H₁₂Br₂N₂NaO₆ (M+Na) cald 472.91, found 472.91.

2,5-dioxopyrrolidin-1-yl3-(2-(2-(2-(3-bromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)propanoate

MS m/z+ for C₁₇H₂₁BrN₂NaO₉ (M+Na) cald 499.04, found 499.04.

2,5-dioxopyrrolidin-1-yl3-(2-(2-(2-(3,4-dibromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)propanoate

MS m/z+ for C₁₇H₂₀Br₂N₂NaO₉ (M+Na) cald 576.95, found 576.95.

Example 17. 4-(2-Pyridyldithio)-4-methylpentanoic acid

4-Mercapto-4-methylpentanoic Acid (Goff, D. et al, Bioconjugate Chem.1990, 1, 381-386) (4.67 g, 31.5 mmol) in methanol (15 ml) was added thesolution of 2,2′-Dithiodipyridine (30.0 g, 136.2 mmol) in the mixture ofmethanol (80 ml) and 100 mM sodium phosphate buffer, pH 7.5 (70 ml).After stirred for 6 h, the mixture was concentrated, extracted withEtOAc/Hexane (1:1). The aqueous solution was adjusted to pH 3 andextracted with EtOAc (3×100 ml). The organic layers were combined, driedover Na₂SO₄, filtered, evaporated and purified on SiO2 column(MeOH/CH2Cl2/HOAc, 1:15:0.01) to afford the title compound (7.05 g,87%). MS m/z+ for C₁₁H₁₆NO₂S₂(M+H) cald 258.05, found 258.05.

Example 18. N-Succinimidyl 4-(2-pyridyldithio)-4-methylpentanoate (243)(SMDP Linker)

4-(2-pyridyldithio)-4-methylpentanoic acid (2.0 g, 7.78 mmol) in CH₂Cl₂(20 ml) was added N-hydroxysuccimide (1.10 g, 9.56 mmol) and EDC (4.0 g,20.8 mmol) and the mixture was stirred overnight, evaporated andpurified on SiO₂ column (EtOAc/CH₂Cl₂, 1:10) to afford the titlecompound (2.48 g, 90%). MS m/z+ for C₁₅H₁₈NaN₂O₄S₂ (M+Na) cald 377.07,found 377.08.

Example 19.(3aR,4R,6S,6aR)-6-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyltetrahydro-furo[3,4-d][1,3]dioxol-4-ol(62)

To a stirred slurry of D-gulonic-lactone (20.01 g, 112.37 mmol) andanhydrous CuSO₄ (25.0 g, 157.22 mmol) in dry acetone (450 mL) was addedcone. H₂SO₄ (1.6 mL), and the mixture was stirred for 24 h at roomtemperature. The pH of the solution was adjusted to 7 with Ca(OH)₂, andthe resulting slurry was filtered and evaporated in vacuo to afford adiacetonide (2,3:5,6-O-diisopropylidene-D-gulono-1,4-lactone) as alight-yellow syrup which was used in the next step without furtherpurification. To a stirred solution of diacetonide in THF (300 mL) at−78° C. was added slowly 1 M solution of DIBAL-H (180 ml, 180 mM) intoluene. After being stirred for 1 h at −78° C., the reaction mixturewas quenched with water (50 mL) and filtered through Celite. The organiclayer was separated, dried (Na₂SO₄), and concentrated in vacuo. Theresidue was purified by silica gel chromatography with hexane-ethylacetate (5:1) to give the title compound (25.27 g, 83% two steps) ascolorless syrup. ESI MS m/z+ for C₁₂H₂₀NaO₆ (M+Na) cald 283.12, found283.12.

Example 20.(3aR,4R,6S,6aR,Z)-6-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyl-N-(2-methylpropylidene)tetrahydrofuro[3,4-d][1,3]dioxol-4-amineoxide (64)

A mixture of 2,3:5,6-O-diisopropylidene-D-gulofuranose (62) (10.0 g,38.4 mmol) and hydroxylamine hydrochloride (25.01 g, 360.87 mmol) inpyridine (150 mL) was stirred at room temperature for 1 h. The reactionmixture was concentrated in vacuo, added water (250 mL) and extractedwith dichloromethane. The combined organic extracts were washed withbrine, dried (MgSO₄), concentrated in vacuo and filtered through shortsilica gel column eluted with ethyl acetate to give2,3:5,6-O-diisopropylidene-D-gulose oxime (63) (10.34 g, 98%) as acolorless vitreous substance which was used directly without furtherpurification. A mixture of this crude oxime (63) (10.30 g, 37.43 mmol),isobutyraldehyde (3.00 g, 41.66 mmol), and MgSO₄ (3 g, 25 mmol) wasstirred at the room temperature overnight. The mixture was filteredthrough a pad of Celite. The filtered was concentrated in vacuo and theresidue was passed through a short SiO₂ column eluted with ethyl acetateto afford the title compound (11.57 g, 94% yield) as white solid. ESI MSm/z+ for C₁₆H₂₇NO₆ cald 329.18. found 329.18.

Example 21.(3R,SR)-2-((3aR,4R,6S,6aR)-6-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-3-[(−)-10′,2′-camphorsultam]-N-propylisoxazolidine-5-carboxamide(65)

A mixture of (3aR,4R,6S,6aR,Z)-6-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyl-N-(2-methylpropylidene)tetrahydrofuro[3,4-d][1,3]dioxol-4-amineoxide (6.00 g 18.22 mmol) and (2R)-N-(acryloyl) bornane-10,2-sultams(5.10 g, 18.95 mmol) in CH₂Cl₂ (50 mL) was heated under reflux for 37 h.After concentration, the residue was recrystallized from EtOH (30 mL) togive the title compound (8.72 g, 80% yield) as a colorless solid. Flashchromatography (silica gel, hexane/AcOEt 7:3) of the mother liquor gavefurther (0.47 g, 4%) of the title compound as a colorless solid. MS ESI:m/z: [M+Na]+, calcd for C29H46N2NaO9S 621.28. Found, 621.28.

Example 22.(3R,SR)-2-(tert-butoxycarbonyl)-3-isopropylisoxazolidine-5-carboxylicacid (67)

A solution of LiOH (5.0 g, 208.7 mmol) in H₂O (60 mL) at 45° C. wasadded to a solution of(3R,5R)-2-((3aR,4R,6S,6aR)-6-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-3-[(−)-10′,2′-camphorsultam]-N-propylisoxazolidine-5-carboxamide(30.0 g, 48.2 mmol) in THF (100 mL) and MeOH (60 mL). After stirred for1 h, the mixture was concentrated, poured into H₂O (150 mL) and themixture was adjusted to pH 9 with 4M HCl aq. The mixture was extractedwith EtOAc, then the aqueous layer was adjusted to pH 3 with 4M HCl, Themixture was extracted with EtOAc, the organic extract was washed withbrine, dried over Na₂SO₄, filtered, and then concentrated in vacuo. Theresidue was triturated with hexane to give(3R,5R)-2-{(3aR,4R,6S,6aR)-6-[(R)-2,2-dimethyl-1,3-dioxolan-4-yl]-2,2-dimethyltetrahydrofuro[3,4-d,1,3]dioxol-4-yl}-3-isopropylisoxazolidine-5-carboxylicacid (66a) (17.1 g 88%) as a colorless solid. This material was used forthe next step without further purification. MS ESI: m/z: [M+Na]+, calcdfor C₁₉H₃₁NNaO₈, 424.19, Found, 424.19. To a solution of this material(8.0 g, 19.95 mmol) in MeCN (80 mL) was added 60% HCl O₄ aq. (6.0 mL,35.77 mmol) at 45° C., and the mixture was stirred at RT for 1 h. Afterconcentration, the residue was dissolved in 1,4-dioxane (40 mL), andthen a suspension of NaHCO₃ (25 g, 297 mmol) in H₂O (32 mL) and Boc₂O(4.80 g, 22.00 mmol) was added at 4° C. The mixture was stirred at RTfor 4 h, concentrated, diluted with H₂O and EtOAc/Hexane (1:1) andseparated. The aqueous layer was adjusted to pH 3 with 4 M HCl aq. Themixture was extracted with EtOAc, the organic layers were washed withbrine, dried over Na₂SO₄, filtered, and evaporated in vacuo. The residuewas triturated with hexane to give(3R,5R)-2-(tert-butoxycarbonyl)-3-isopropylisoxazolidine-5-carboxylicacid (4.91 g, 95%) as a colorless amorphous solid. MS ESI: m/z: [M+Na]+,calcd for C₁₂H₂₁NNaO₅, 282.13, Found, 282.13.

Example 23. Methyl(R)-2-[(3R,SR)-2-[(t-butyl-yl)methoxycarbonyl]-3-isopropylisoxazolidine-5-carboxamido]-3-(triphenylmethylthio)propionate(68)

iPr2NEt (0.75 mL, 4.31 mmol) and TBTU (2.50 g, 7.78 mmol) were added toa solution of(3R,5R)-2-(tert-butoxycarbonyl)-3-isopropylisoxazolidine-5-carboxylicacid (1.01 g, 3.89 mmol) and 1-(S)-Tr-cysteine methyl esterhydrochloride (1.76 g, 4.27 mmol) in CH₂Cl₂ (15 mL) at 4° C. and themixture was stirred at RT for overnight. The mixture was poured intoNaHCO₃ (sat.) solution, extracted with CH₂Cl₂, dried over Na₂SO₄,filtered, concentrated and silica gel chromatography (hexane/AcOEt 1:2)to give the title compound (2.10 g, 87%) as colorless amorphous solid.MS ESI: m/z: [M+Na]+, calcd for C₃₅H₄₂N₂NaO₆S, 641.27, Found, 641.26.

Example 24. (3R,SR)-tert-butyl3-isopropyl-5-(4-(methoxycarbonyl)thiazol-2-yl)isoxazolidine-2-carboxylate(69)

Tf₂O (2.0 mL, 12.0 mmol) was added to a solution of Ph₃P═O (4.10 g,14.74 mmol) in CH₂Cl₂ (40.0 mL) and the mixture was stirred at −10° C.for 1 h. A solution of Methyl(R)-2-[(3R,5R)-2-[(t-butyl-yl)methoxycarbonyl]-3-isopropylisoxazolidine-5-carboxamido]-3-(triphenylmethylthio)propionate(68) (4.00 g, 6.47 mmol) in CH₂Cl₂ (20 mL) was added to the reactionmixture at −10° C. and the mixture was stirred at RT for 6 h, added toNaHCO₃ saturated solution at 4° C. and extracted with CH₂Cl₂. Theorganic extracts were washed with brine, dried over Na₂SO₄, filtered,concentrated in vacuo and SiO₂ chromatography (hexane/AcOEt 3:2) to givethe corresponding thiazolidine derivative as a yellow amorphous solid.MnO₂ (5.80 g, 66.7 mmol) was added to a solution of this material inCH₂Cl₂ (60 mL) and the mixture was stirred at RT for 24 h. The mixturewas filtrated via Celite, concentrated in vacuo and SiO₂ chromatography(hexane/AcOEt 3:2) to give the title compound (69) (1.75 g, 75%) as acolorless amorphous solid. ESI: m/z: [M+Na]+, calcd for C₁₆H₂₄N2NaO₅S,379.13, Found, 379.14.

Example 25. Methyl2-((1R,3R)-3-(tert-butoxycarbonylamino)-1-hydroxy-4-methylpentyl)-thiazole-4-carboxylate(70)

Mo(CO)₆ (1.10 g, 3.12 mmol) was added to a solution of(3R,5R)-tert-butyl3-isopropyl-5-(4-(methoxycarbonyl)thiazol-2-yl)isoxazolidine-2-carboxylate(69) (1.00 g, 2.81 mmol) in CH₃CN (20 ml) and H₂O (2 ml) and the mixturewas stirred at 70° C. for 16 h. After concentration, the residue wasdiluted with EtOAc (50 mL) and a 10% aq. solution of citric acid (50ml). NaIO₄ was added to the mixture until the aqueous layer becameclear, and extracted with EtOAc. The organic extracts were washed with a10% aq. Na₂S₂O₃ and brine, dried over Na₂SO₄, filtered, concentrated andSiO₂ chromatography (hexane/EtOAc 3:2) to give the title compound (906mg, 90%) as a colorless solid. MS ESI: m/z: [M+Na]+, calcd forC₁₆H₂₆N₂NaO₅S, 381.14, Found, 381.14.

Example 26. Methyl2-((1R,3R)-1-acetoxy-3-(tert-butoxycarbonyl-amino)-4-methylpentyl)-thiazole-4-carboxylate(71)

Compound 70 (900 mg, 2.51 mmol) in pyridine (15 ml) was added Ac₂O (0.5ml, 5.29 mmol) and the mixture was stirred overnight, concentrated, SiO₂chromatography (hexane/EtOAc 4:1) to give the title compound (950 mg,95%) as a colorless solid. MS ESI: m/z: [M+Na]+, calcd forC₁₈H₂₈N2NaO₆S, 423.15, Found, 423.16.

Example 27.2-((1R,3R)-1-acetoxy-3-(tert-butoxycarbonylamino)-4-methylpentyl)thiazole-4-carboxylicacid (72)

Compound 71 (940 mg, 2.35 mmol) in THF (15 ml) was added NaH (120 mg,3.0 mmol, 60% in oil) at 4° C. and the mixture was stirred for 2 h, thenCH₃I (0.155 ml, 2.49 mmol) was added. After stirred overnight,concentrated, re-dissolved in EtOAc, filtered through short SiO₂ columnand evaporated to dryness to give a crude2-((1R,3R)-1-acetoxy-3-(tert-butoxycarbonyl-(methyl)amino)-4-methylpentyl)thiazole-4-carboxylicacid (73a). To the dryness compound (73a) in 1,2-dichloroethane (20 ml)was added Trimethyltin hydroxide (620 mg, 3.43 mmol). The mixture wasstirred at 80° C. overnight, concentrated, re-dissolved MeOH/CH₂Cl₂/HOAc(1:5:0.01, 20 ml), filtered through short SiO₂ column, concentrated andcoevaporated with toluene to a dryness. To the dryness compound inpyridine (15 ml) was added Ac₂O (0.4 ml, 4.23 mmol). After stirredovernight, the mixture was concentrated, SiO₂ chromatography(MeOH/CH₂Cl₂/HOAc (1:10:0.01) to give the title compound (735 mg, 78%)as a colorless solid. ESI: m/z: [M+Na]+, calcd for C₁₈H₂₈N2NaO₆S,423.15, Found, 423.16.

Example 28. Methyl2-((1R,3R)-3-(tert-butoxycarbonyl(methyl)amino)-1-(3-(1,3-dioxoisoindolin-2-yl)propoxy)-4-methylpentyl)thiazole-4-carboxylate(86)

Compound 70 (850 mg, 2.37 mmol) in THF (15 ml) at −20 OC was added NaH(100 mg, 2.5 mmol, 60% in oil). After stirred for 30 min at −20 OC,N-(3-Bromopropyl)phthalimide (655 mg, 2.4 mmol) was added and themixture was stirred at −20 OC for 30 min and then warmed up to roomtemperature in 4 h. The reaction mixture was quenched with methanol (0.5ml), diluted with CH₂Cl₂ (60 ml), filtered through a short silica gelcolumn, evaporated to dryness to provide crude methyl2-((1R,3R)-3-(tert-butoxycarbonyl-amino)-1-(3-(1,3-dioxoisoindolin-2-yl)propoxy)-4-methylpentyl)thiazole-4-carboxylate85 which was used directly without further purification. To the crudecompound 85 in THF (25 ml) at room temperature was added NaH (170 mg,4.25 mmol, 60% in oil). After stirred for 45 min, CH₃I (0.20 ml, 3.21mmol) was added. The mixture was stirred at room temperature overnight,quenched with NaH₂PO₄ (2.0 M, 2 ml). The mixture was added DMA (5 ml),evaporated in vacuo, SiO₂ chromatography with EtOAc/CH₂Cl₂ (1:10-1:4) toafford the title compound (921 mg, 69%). ESI: m/z: [M+Na]+, calcd forC₂₈H₃₇N3NaO₇S, 582.22, Found, 582.22.

Example 29.2-((1R,3R)-3-(tert-butoxycarbonyl(methyl)amino)-1-(3-(1,3-dioxoisoindolin-2-yl)propoxy)-4-methylpentyl)thiazole-4-carboxylicacid (87)

To the dryness compound 86 (910 mg, 1.63 mmol) in 1,2-dichloroethane (20ml) was added Trimethyltin hydroxide (400 mg, 2.21 mmol). The mixturewas stirred at 80° C. overnight, concentrated, purified on SiO₂ columneluted with CH₃OH/CH₂Cl₂/HOAc (1:10:0.01) to afford the title compound(756 mg, 85%). ESI: m/z: [M+Na]+, calcd for C₂₇H₃₆N3NaO₇S, 546.22.Found, 546.22.

Example 30. Methyl2-((1R,3R)-1-acetoxy-3-(tert-butoxycarbonyl(3-(1,3-dioxoisoindolin-2-yl)propyl)amino)-4-methylpentyl)thiazole-4-carboxylate(89)

To the compound 71 (800 mg, 2.00 mmol) in THF (30 ml) at roomtemperature was added NaH (150 mg, 3.75 mmol, 60% in oil). After stirredfor 45 min, N-(3-Bromopropyl)-phthalimide (655 mg, 2.4 mmol) was added.The mixture was stirred at room temperature overnight, quenched withNaH₂PO₄ (2.0 M, 2 ml). The mixture was added DMA (5 ml), evaporated andpurified on SiO₂ column eluted with EtOAc/CH₂Cl₂ (1:10˜1:4) to affordthe title compound (971 mg, 82%). ESI: m/z: [M+Na]+, calcd forC₂₉H₃₇N3NaO₈S, 610.22, Found, 610.22.

Example 31.2-((1R,3R)-1-acetoxy-3-(tert-butoxycarbonyl(3-(1,3-dioxoisoindolin-2-yl)propyl)amino)-4-methylpentyl)thiazole-4-carboxylicacid (90)

To the dryness compound (89) (900 mg, 1.53 mmol) in 1,2-dichloroethane(35 ml) was added trimethyltin hydroxide (400 mg, 2.21 mmol). Themixture was stirred at 80° C. overnight, concentrated. Then the mixturein pyridine (20 ml) was added Ac₂O (3 ml) and stirred overnight,evaporated, purified on SiO₂ column (MeOH/CH₂Cl₂/HOAc, 1:10:0.01) toafford the title compound (755 mg, 86%). ESI: m/z: [M+Na]+, calcd forC₂₈H₃₅N₃NaO₈S, 596.20, Found, 596.20.

Example 32. (S)-Ethyl5-(4-(benzyloxy)phenyl)-4-(tert-butoxy-carbonylamino)-2-methylpent-2-enoate(185)

(S)-Methyl3-(4-(benzyloxy)phenyl)-2-(tert-butoxy-carbonylamino)propanoate 184(8.00 g, 20.76 mmol) in CH₂Cl₂ (250 ml) at −78° C. was added dropwiseDIBAL (40 ml, 40 mmol, 1.0 M) in CH₂Cl₂. After stirred at −78° C. for 2h, the reaction was quenched with addition of MeOH (5 ml). The mixturewas warmed to RT, acidified with 1 M HCl to pH 4 and separated. Theaqueous layer was extracted with DCM (2×150 ml). The organic layers werewashed with water, dried over Na₂SO₄, filtered and evaporated to drynessto form cude aldehyde intermediate. Then the crude intermediate aldehydewas dissolved in DCM, the ylide solution prepared from1-(1-ethoxycarbonyl ethyl)-triphenylphosphonium bromide (18.0 g, 40.64mmol) and KOtBu (5.00 g, 44.64 mmol) in CH₂Cl₂ (80 ml) at RT was addedat. After stirred at RT over night, the mixture was extractedconcentrated and purified by SiO₂ chromatography (EtOAc/Hexane, 1:8˜1:4)to afford (6.90 g, 76%) of the title compound. ESI: m/z: [M+Na]+, calcdfor C₂₆H₃₃NNaO₅, 462.22, Found, 462.22.

Example 33. (4R)-ethyl4-((tert-butoxycarbonyl)amino)-5-(4-hydroxyphenyl)-2-methylpentanoate(186)

(S)-Ethyl 5-(4-(benzy loxy)phenyl)-4-(tert-butoxy-carbonylamina)-2-methylpent-2-enoate (185) (6.70 g, 15.26 mmol) in ahydrogenation bottle was charged methanol (150 ml), Pd/C (0.3 g, 10%Pd). The hydrogenation reaction was conducted at 30 psi for 6 h. Themixture was filtered through Celite, evaporated and crystallized withEtOH/hexane to the title compound (186) (4.61 g, 86% yield). ESI: m/z:[M+Na]+, calcd for C₁₉H₂₉NNaO₅, 374.20, Found, 374.30.

Example 34. (4R)-ethyl4-((tert-butoxycarbonyl)amino)-5-(4-hydroxy-3-nitrophenyl)-2-methylpentanoate(187)

To a solution of compound 186 (4.50 g, 12.81 mmol) in anhydrous CH₂Cl₂(200 ml) was added Ac₂O (2 ml, 21.16 mmol) and fuming HNO₃ (0.65 ml,14.07 mmol). The mixture was stirred at RT for 4 h, diluted with water(150 ml), separated and the aqueous layer was extracted with EtOAc. Theorganic layers were combined, dried over Na₂SO₄, evaporated and purifiedon SiO₂ column (EtOAc/DCM, 1:10) to afford (4.21 g, 83%) of the titlecompound. ESI: m/z: [M+Na]+, calcd for C₁₉H₂₈N₂NaO₇, 419.19, Found,419.20.

Example 35. ethyl4-((tert-butoxycarbonyl)amino)-2-methyl-5-(3-nitro-4-(phosphonooxy)phenyl)pentanoate(188)

Compound 187 (4.00 g, 10.09 mmol) in a mixture of CH₃CN (70 ml) and DMA(30 ml) was added DIPEA (4.00 ml, 23.00 mmol) at 0° C. After stirred for2 min, POCl₃ (2.00 ml, 21.45 mmol) was added dropwise at 0° C. Themixture was stirred at RT for 8 h, and quenched with slowly addition ofNaHCO₃ (3.5 g, 41.60 mmol) in water (20 ml) at 0° C. After stirred at 0°C. overnight, the mixture was concentrated and purified on C-18cartridge (20×4 cm) eluted with gradient mixture, 25 ml/min, A: 0.5%HOAc, B: CH₃OH, from 100% A in 10 min, then to 75% A and 25% B in 45min. The fractions containing the product was pooled and evaporated toafford the title compound (3.89 g, 81% yield). ESI: m/z: [M−H]⁻, calcdfor C₁₉H₂₈N₂O₁₀P, 475.16, Found, 475.20.

Example 36.(4R)-4-((tert-butoxycarbonyl)amino)-2-methyl-5-(3-nitro-4-(phosphonooxy)phenyl)pentanoicacid (189)

To a solution of LiOH (5.0 g, 208.7 mmol) in H₂O (60 mL) was added to asolution of compound (188) (3.75 g, 7.87 mmol) in THF (100 mL). Afterstirred for 4 h at 0° C., the mixture was was adjusted to pH ˜6 with 4MHCl, concentrated, and purified by C-18 chromatography eluted withgradient mixture, 25 ml/min, A: 0.5% HOAc, B: CH₃OH, from 100% A in 10min, then to 75% A and 25% Bin 45 min. The fractions containing theproduct was pooled and evaporated to afford the title compound (2.82 g,80% yield). ESI: m/z: [M−H]⁻, calcd for C₁₇H₂₄N₂O₁₀P, 447.12, Found,447.20.

Example 37.(4R)-5-(3-amino-4-(phosphonooxy)phenyl)-4-((tert-butoxycarbonyl)amino)-2-methylpentanoicacid (190)

Compound (189) (2.60 g, 5.80 mmol) in a hydrogenation bottle was chargedmethanol (80 ml), Pd/C (0.2 g, 10% Pd). The hydrogenation reaction wasconducted at 35 psi of H₂ for 6 h. The mixture was filtered throughCelite, evaporated to afford crude title compound (190) (2.18 g, 90%yield), which was used directly without further purification. ESI: m/z:[M+Na]+, calcd for C₁₇H₂₆N₂O₈P, 417.15, Found, 417.15.

Example 38. (S)-methyl2-((tert-butoxycarbonyl)amino)-3-(4-hydroxy-3-nitrophenyl)propanoate(196)

To a solution of (S)-methyl2-((tert-butoxycarbonyl)amino)-3-(4-hydroxyphenyl) propanoate (195) (4.5g, 15.24 mmol) in anhydrous CH₂Cl₂ (240 ml) was added Ac₂O (4 ml, 42.32mmol) and fuming HNO₃ (0.85 ml, 18.40 mmol). The mixture was stirred atRT for 4 h, diluted with water (150 ml), separated and the aqueous layerwas extracted with EtOAc. The organic layers were combined, dried overNa₂SO₄, evaporated and purified on SiO₂ column (EtOAc/DCM, 1:10) toafford (4.30 g, 83%) of the title compound. ESI: m/z: [M+Na]+, calcd forC₁₅H₂₀N2NaO₇, 363.13, Found, 363.20.

Example 39. (S)-methyl2-((tert-butoxycarbonyl)amino)-3-(3-nitro-4-(phosphonooxy)phenyl)propanoate(197)

To a solution of compound 196 (4.10 g, 12.05 mmol) in a mixture of CH₃CN(90 ml) was added DIPEA (4.00 ml, 23.00 mmol) at 0° C. After stirred for2 min, POCl₃ (2.00 ml, 21.45 mmol) was added dropwise at 0° C. Themixture was stirred at RT for 8 h, and quenched with slowly addition ofNaHCO₃ (3.5 g, 41.60 mmol) in water (20 ml) at 0° C. After stirred at 0°C. overnight, the mixture was concentrated and purified on C-18cartridge (20×4 cm) eluted with gradient mixture, 25 ml/min, A: 0.5%HOAc, B: CH₃OH, from 100% A in 10 min, then to 75% A and 25% Bin 45 min.The fractions containing the product was pooled and evaporated to afford(4.20 g, 83%) the title compound. ESI: m/z: [M−H]⁻, calcd forC₁₅H₂₀N₂O₁₀P, 419.08. Found, 419.10.

Example 40.3-[3-amino-4-(phosphonooxy)phenyl]-(2R)-2-{[(tert-butoxy)carbonyl]amino}-propanoicacid (198)

To the dryness compound (197) (4.0 g, 9.52 mmol) in the mixture of1,2-dichloroethane (50 ml) and DMA (60 ml) was added trimethyltinhydroxide (4.00 g, 22.1 mmol). The mixture was stirred at 80° C. for 6h, evaporated, filtered through short SiO₂ column eluted with water/MeCN(1:4). The fractions containing the product were pooled, concentrated togenerate(S)-2-((tert-butoxycarbonyl)amino)-3-(3-nitro-4-(phosphonooxy)phenyl)propanoicacid. In a hydrogenation bottle was charged DMA (70 ml), Pd/C (0.3 g,10% Pd), followed by the addition of the prepared propanoic acid. Thehydrogenation reaction was conducted at 30 psi of hydrogen for 6 h. Themixture was filtered through Celite, evaporated, and crystallized toafford (2.86 g, 80% yield) of the title compound (198), which was useddirectly without further purification. ESI: m/z: [M−H]⁻, calcd forC₁₄H₂₀N₂O₈P, 375.10, Found, 375.10.

Example 41. Benzyl3-[4-(benzyloxy)phenyl]-(2R)-2-{[(tert-butoxy)carbonyl]-(methyl)-amino}-propanoate(200)

A solution of benzyl3-[4-(benzyloxy)phenyl]-(2R)-2-{[(tert-butoxy)carbonyl]-amino}propanoate(4.0 g, 8.67 mmol) in THF (60 ml) was added NaH (430 mg, 10.75 mmol, 60%in oil). After stirred at RT for 1 h, CH₃I (1.82 g, 12.82 mmol) wasadded, and the mixture was stirred overnight, quenched with CH₃OH (0.5ml), evaporated and purified on SiO2 column (EtOAc/CH₂Cl₂, 1:10) toafford the title compound (3.83 g, 93%). MS ESI: m/z: [M+Na]+, calcd forC₂₉H₃₃NNaO₅, 498.24, Found, 498.24.

Example 42.(2R)-2-{[(tert-butoxy)carbonyl](methyl)amino}-3-(4-hydroxy-3-nitrophenyl)propanoicacid (201)

Compound 200 (3.80 g, 8.00 mmol) in a hydrogenation bottle was chargedmethanol (80 ml), Pd/C (0.3 g, 10% Pd). The hydrogenation reaction wasconducted at 30 psi of hydrogen for 6 h. The mixture was filteredthrough Celite, evaporated to afford crude(2R)-2-{[(tert-butoxy)carbonyl](methyl)amino}-3-(4-hydroxyphenyl)propanoicacid (201a), which was used directly without further purification. Tothe compound 201a in anhydrous CH₂Cl₂ (240 ml) at −25° C. was addeddropwise a mixture of SnCl₄ (1.5 ml, 12.75 mmol) and fuming HNO₃ (0.60ml, 12.98 mmol) in CH₂Cl₂ (40 ml). The mixture was stirred at −25° C.for 75 min, quenched with saturated NaHCO₃ to pH 3-4, separated and theaq. layer was extracted with EtOAc. The organic layers were combined,dried over Na₂SO₄, concentrated and purified on SiO₂ column(MeOH/DCM/HOAc 1:8:0.01) to afford (1.98 g, 73%) of the title compound.ESI: m/z: [M+Na]+, calcd for C₁₅H₂₀N2NaO₇, 363.13, Found, 363.13.

Example 43.(2R)-2-{[(tert-butoxy)carbonyl](methyl)amino}-3-[3-nitro-4-(phosphonooxy)-phenyl]propanoicacid (202)

To a solution of compound 201 (1.98 g, 5.82 mmol) in a mixture of CH₃CN(30 ml) and DMA (30 ml) was added DIPEA (2.00 ml, 11.50 mmol) at 0° C.After stirred for 2 min, POCl₃ (1.10 ml, 11.79 mmol) was added dropwiseat 0° C. The mixture was stirred at RT for 8 h, and quenched with slowlyaddition of NaHCO₃ (2.0 g, 23.80 mmol) in water (10 ml) at 0° C. Afterstirred at 0° C. overnight, the mixture was concentrated and purified onC-18 cartridge (20×4 cm) eluted with gradient mixture, 25 ml/min, A:0.5% HOAc, B: CH₃OH, from 100% A in 10 min, then to 75% A and 25% Bin 45min. The fractions containing the product was pooled and evaporated toafford (1.96, 80%) the title compound. ESI: m/z: [M−H], calcd forC₁₅H₂₀N₂O₁₀P, 419.09, Found, 419.09.

Example 44.3-[3-amino-4-(phosphonooxy)phenyl]-(2R)-2-[(tert-butoxycarbonyl)(methyl)-amino]-propanoicacid (203)

Compound 202 (1.96 g, 4.67 mmol) in a hydrogenation bottle was chargedDMA (60 ml), Pd/C (0.2 g, 10% Pd). The hydrogenation reaction wasconducted at 30 psi of hydrogen for 6 h. The mixture was filteredthrough Celite, evaporated to dryness to afford (1.74 g, 95%) of thetitle compound (203), which was used directly without furtherpurification. ESI: m/z: [M−H]⁻, calcd for C₁₅H₂₂N₂O₈P, 389.12, Found,389.12.

Example 45. Tert-butyl N-(1-oxo-1-phenylpropan-2-(2R)-yl)carbamate (204)

(1S,2R)-(+)-Norephedrine (7.0 g, 46.29 mmol) in the mixture of THF (40ml) and 1M NaHCO₃ (100 ml) at 4° C. was added dropwise Boc₂O (10.15 g,46.53 mmol) in THF (60 ml) in 45 min. The mixture then stirred at RT for6 h, concentrated, extracted with EtOAc, dried over Na₂SO₄, concentratedand filtered through short SiO₂ column eluted with EtOAc/Hexane (1:2),concentrated to afford the crude tert-butylN-((1S)-hydroxy-1-phenylpropan-2-(2R)-yl)carbamate (204b) (10.81, 93%).MS ESI: m/z+: [M+Na]⁺, calcd for C₁₄H₂₁NaNO₃, 274.15, Found, 274.15. Thecrude compound was used directly without further purification. Thecompound (204b) in CH₂Cl₂ (50 ml) was added Dess-Martin periodinanesolution in CH₂Cl₂ (180 ml, 0.3 M). After stirred for 1 h, the mixturewas added ice cold NaOH (1 M, 100 ml), separated and the organic layerswere washed with 1M NaH₂PO₄, pH 6 (100 ml), dried over Na₂SO₄,evaporated and purified on SiO2 column (EtOAc/hexane 1:5) to afford thetitle compound 204 (9.34 g, 81% in two steps). MS ESI: m/z+: [M+Na]⁺,calcd for C₁₄H₁₉NaNO₃, 272.14, Found, 272.14.

Example 46.(1R,3R)-3-((2S,3S)-N-(methyl)-3-methyl-2-((R)-1-methylpiperidine-2-carboxamido)-pentamido)-4-methyl-1-(4-((1-oxo-1-phenylpropan-2-yl)carbamoyl)thiazol-2-yl)-pentylacetate (205)

Compound 204 (180 mg, 0.722 mmol) in 4 ml of dioxane was added HCl conc.(1.0 ml, 37%) at 4° C. and the mixture was stirred at RT for 30 min,evaporated and coevaporated with toluene to dryness. Then to the drynesssolid in DMA (7 ml) were added compound 106 (251 mg, 0.466 mmol), EDC(305 mg, 1.56 mmol) and DIPEA (0.13 ml, 0.747 mmol) and the mixture wasstirred for 8 h, evaporated and purified on SiO₂ column (EtOAc/CH₂Cl₂,1:4) to afford the title compound 205 (255.3 mg, 82%). ESI: m/z+:[M+Na]+, calcd for C₃₅H₅₁NaN₅O₆S, 692.36. Found, 692.36.

Example 47.(1R,3R)-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)-pentanamido)-1-(4-((R)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-12-oxo-15-phenyl-3,6,9-trioxa-13,14-diazaheptadec-14-en-16-ylcarbamoyl)thiazol-2-yl)-4-methylpentylacetate (206)

Compound 205 (75 mg, 0.112 mmol) in methanol (5 ml) was added compound12 (50 mg in HCl salt, 0.126 mmol) and HOAc (3 ul, 0.052 mmol). Themixture was stirred overnight, neutralized with DIPEA (23 ul, 0.132mmol), evaporated and purified on SiO₂ cartridge (4 g, EtOAc/CH₂Cl₂,1:5-1:3) to afford the title compound 206 (79.3 mg, 70%). MS ESI: m/z+:[M+Na]+, calcd for C₅₀H₇₄NaN₈O₁₂S, 1033.51, Found, 1033.50.

Example 48.(1S,2R)-2-(2-((1R,3R)-1-acetoxy-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-1-phenyl-propyl3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)-propanoate(211)

Compound 208a (95 mg, 0.141 mmol) and3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)propanoicacid (55 mg, 0.182 mmol) in CH₂Cl₅ (5 ml) were added DCC (122 mg, 0.591mmol) and DMAP (25 mg, 0.204 mmol) and the mixture was stirredovernight, evaporated and purified with SiO₂ chromatography(EtOAc/CH₂Cl₂, 1:3) to afford the title compound (95.1 mg, 71%). MS ESI:m/z+: [M+Na]⁺, calcd for C₄₈H₇₀NaN₆O₁₂S, 977.47. Found, 977.47.

Example 49. 2,5-dioxopyrrolidin-1-yl2-((1R,3R)-1-acetoxy-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxylate(234)

Compound 106 (788.1 mg, 1.464 mmol) in DMF (10 ml) were added NHS (202.0mg, 1.756 mmol) and EDC (980 mg, 5.104 mmol) and the mixture was stirredovernight, evaporated and purified with SiO₂ chromatography(EtOAc/CH₂Cl₂, 1:3) to afford the title compound (762.8 mg, 82%). MSESI: m/z+: [M+Na]⁺, calcd for C₃₀H₄₅NaN₅O₈S, 658.30, Found, 658.30.

Example 50.(4R)-4-(tert-butoxycarbonylamino)-5-(3-(5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentanamido)-4-(phosphonooxy)phenyl)-2-methylpentanoicacid (235)

Compound 190 (825.1 mg, 1.973 mmol) in DMF (7 ml) was added2,5-dioxopyrrolidin-1-yl5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentanoate (23d) (711 mg, 2.417mmol) and DIPEA (0.250 ml, 1.438 mmol), and the mixture was stirredovernight, evaporated and purified with C-18 chromatography (4×25 cm,v=15 ml/min, 100% of 1% HOAc to 75% of 1% HOAc/25% MeOH in 45 min) toafford the title compound 235 (895.7 mg, 76%). MS ESI: m/z−: [M−H]⁻,calcd for C₂₆H₃₅N30₁₁P, 596.21, Found, 596.21.

Example 51.(4R)-4-(tert-butoxycarbonylamino)-5-(3-(3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)propanamido)-4-(phosphonooxy)phenyl)-2-methylpentanoicacid (236)

Compound 190 (632.5 mg, 1.512 mmol) in DMF (7 ml) were added2,5-dioxopyrrolidin-1-yl3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)propanoate(24c) (727 mg, 1.826 mmol) and DIPEA (0.250 ml, 1.438 mmol), and themixture was stirred overnight, evaporated and purified with C-18chromatography (4×25 cm, v=15 ml/min, 100% of 1% HOAc to 75% of 1%HOAc/25% MeOH in 45 min) to afford the title compound 236 (763.2 mg,72%). MS ESI: m/z−: [M−H]⁻, calcd for C₃₀H₄₄N₃O₁₄P, 700.25, Found,700.25.

Example 52.(4R)-4-(2-((1R,3R)-1-acetoxy-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-5-(3-(5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentanamido)-4-(phosphonooxy)phenyl)-2-methylpentanoicacid (239)

Compound 235 (102 mg, 0.171 mmol) in 1,4-dioxane (4 ml) was added conc.HCl (1 ml, 37%) and the mixture was stirred for 30 min, evaporated todryness to afford the crude compound 237. To the crude compound in DMA(5 ml) were added compound 234 (110 mg, 0.173 mmol) and DIPEA (30 ul,0.172 mmol), and the mixture was stirred for overnight, evaporated andpurified by SiO₂ chromatography (1% HOAc in water/acetone, 1:9-1:4) toafford the title compound 239 (123.2 mg, 71%). MS ESI: m/z−: [M−H]⁻,calcd for C₄₇H₆₇N₇O₁₄PS, 1016.42, Found, 1016.42.

Example 53.(4R)-4-(2-((1R,3R)-1-acetoxy-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-5-(3-(3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)-propanamido)-4-(phosphonooxy)phenyl)-2-methylpentanoicacid (240)

Compound 236 (108 mg, 0.154 mmol) in 1,4-dioxane (4 ml) was added conc.HCl (1 ml, 37%) and the mixture was stirred for 30 min, evaporated todryness to afford the crude compound 238. To the crude compound in DMA(5 ml) were added compound 234 (110 mg, 0.173 mmol) and DIPEA (30 ul,0.172 mmol), and the mixture was stirred for overnight, evaporated andpurified by SiO₂ chromatography (1% HOAc in water/acetone, 1:9-1:4) toafford the title compound 240 (131.2 mg, 76%). MS ESI: m/z−: [M−H]⁻,calcd for C₅₁H₇₅N₇O₁₇PS, 1120.47, Found, 1120.48.

Example 54.(4R)-4-(tert-butoxycarbonylamino)-2-methyl-5-(4-(phosphonooxy)-3-(4-(pyridin-2-yldisulfanyl)butanamido)phenyl)pentanoicacid (244)

To a solution of compound 190 (548.3 mg, 1.311 mmol) in DMF (10 ml) wereadded succinimidyl 4-(pyridin-2-yl)disulfanyl)-butyrate (550.2 mg, 1.687mmol) and DIPEA (0.18 ml, 1.03 mmol). The mixture was stirred overnight,evaporated and purified by SiO₂ chromatography (1% HOAc inwater/acetone, 1:9-1:4) to afford the title compound 244 (660.2 mg,80%). MS ESI: m/z−: [M−H]⁻, calcd for C₂₆H₃₆N₃O₉PS₂, 628.16, Found,628.16.

Example 55.(4R)-4-(2-((1R,3R)-1-acetoxy-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-(4-(phosphonooxy)-3-(4-(pyridin-2-yldisulfanyl)butanamido)phenyl)pentanoicacid (248)

Compound 244 (110.5 mg, 0.175 mmol) in 1,4-dioxane (4 ml) was addedconc. HCl (1 ml, 37%) and the mixture was stirred at 4° C. for 30 min,evaporated to dryness to afford the crude compound 246. To the crudecompound in DMA (5 ml) were added compound 234 (110 mg, 0.173 mmol) andDIPEA (30 ul, 0.172 mmol). The mixture was stirred for overnight,evaporated and purified by SiO₂ chromatography (1% HOAc inwater/acetone, 1:9-1:4) to afford the title compound 248 (129.1 mg,71%). MS ESI: m/z−: [M−H]⁻, calcd for C₄₇H₆₈N₇O₁₂PS₃, 1048.38. Found,1048.38.

Example 56.(4R)-4-(2-((1R,3R)-1-acetoxy-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-5-(3-(4-mercaptobutanamido)-4-(phosphonooxy)phenyl)-2-methylpentanoicacid (248b)

Compound 248 (30 mg, 0.0285 mmol) in a mixture of DMA (2 ml) and NaH₂PO₄(0.1 M, pH 7) was added dithiothreitol (20 mg, 0.129 mmol). The mixturewas stirred for 2 h, evaporated and purified by SiO₂ chromatography (1%HOAc in water/CH₃CN, 1:9˜1:4) to afford the title compound 248b (22 mg,85%). MS ESI: m/z−: [M−H]⁻, calcd for C₄₂H₆₄N₆O₁₂PS₂, 939.38. Found,939.38.

Example 57. 4-(4-bromobutyl)-10-oxa-4-azatricyclo[S.2.1.0{circumflexover ( )}{2,6}]dec-8-ene-3,5-dione (271)

10-oxa-4-azatricyclo[5.2.1.0{circumflex over( )}{2,6}]dec-8-ene-3,5-dione (6.0 g, 36.35 mmol) and NaH (1.50 g, 37.50mmol, 60% in oil) were stirred in DMA (60 ml) for 1 h, then1,4-dibromobutane (35.0 g, 162.10 mmol) and NaI (0.50 g, 3.33 mmol) wereadded. The mixture was stirred overnight, quenched with CH₃OH (0.5 ml),evaporated, purified on SiO₂ column (EtOAc/Hexane, 1:8) to afford thetitle compound (9.34 g, 86%). MS ESI: m/z+: [M+Na]⁺, calcd forC₁₂H₁₄BrNaNO₃, 322.02, Found, 322.02.

Example 58. Methyl2-((1R,3R)-3-(tert-butoxycarbonylamino)-1-[4′-(3″,6″-endoxo-Δ-tetrahydrophthalimido)butyloxy]-4-methylpentyl)thiazole-4-carboxylate(272)

Methyl2-((1R,3R)-3-(tert-butoxycarbonylamino)-1-hydroxy-4-methylpentyl)thiazole-4-carboxylate(70) (1.0 g, 2.79 mmol) and NaH (120 mg, 3.00 mmol, 60% in oil) werestirred in THF (30 ml) for 20 min and compound 271 (1.00 g, 3.34 mmol)and NaI (50 mg, 0.33 mmol) were added. The mixture was stirredovernight, quenched with MeOH (0.5 ml), evaporated and purified on SiO₂column (EtOAc/CHCl₂, 1:10) to afford the title compound (1.36 g, 84%).MS ESI: m/z+: [M+Na]+, calcd for C₂₈H₃₉NaN₃O₈S, 600.25, Found, 600.25.

Example 59. Methyl2-((1R,3R)-3-(N,N-tert-butoxycarbonylmethylamino)-1-4′-(3″,6″-endoxo-Δ-tetrahydrophthalimido)butyloxy]-4-methylpentyl)thiazole-4-carboxylate(273)

Compound 272 (1.30 g, 2.25 mmol) and NaH (108 mg, 2.70 mmol, 60% in oil)stirred in DMF (80 ml) for 1 h was added CH₃I (460 mg, 3.24 mmol). Themixture was stirred overnight, evaporated and purified on SiO₂ column(EtOAc/CH₂Cl₂, 1:12-1:8) to afford the title compound (1.01 g, 76%). MSESI: m/z+: [M+Na]⁺, calcd for C₂₉H₄₁NaN₃O₈S, 614.26, Found, 614.26.

Example 60.2-((1R,3R)-3-(N,N-tert-butoxycarbonylmethyl-amino)-1-(4′-maleimido-butyloxy)-4-methylpentyl)thiazole-4-carboxylicacid (274)

To the dryness compound (273) (900 mg, 1.52 mmol) in a mixture of1,2-dichloroethane (30 ml) and toluene was added trimethyltin hydroxide(400 mg, 2.21 mmol). The mixture was stirred at 100° C. overnight,concentrated, purified on SiO₂ column eluted with MeOH/CH₂Cl₂/HOAc(1:10:0.01) to afford the title compound (730 mg, 94%). ESI: m/z:[M+Na]+, calcd for C₂₄H₃₅N₃NaO₇S, 532.22, Found, 532.22.

Example 61. Methyl2-((1R,3R)-1-acetoxy-3-(N,N-(tert-butoxycarbonyl)(4′-(3″,6″-endoxo-Δ-tetrahydrophthalimido)butyl)amino)-4-methylpentyl)-thiazole-4-carboxylate(275)

Compound 71 (1.50 g, 3.74 mmol) and NaH (180 mg, 4.50 mmol, 60% in oil)stirred in DMF (80 ml) for 1 h was added compound 271 (1.48 g, 4.94mmol) and NaI (70 mg, 0.467 mmol). The mixture was stirred overnight,evaporated and purified on SiO₂ column (EtOAc/CH₂Cl₂, 1:10˜1:6) toafford the title compound (1.60 g, 69%). MS ESI: m/z+: [M+Na]⁺, calcdfor C₃₀H₄₁NaN₃O₉S, 642.26, Found, 642.26.

Example 62.2-((1R,3R)-1-acetoxy-3-(N,N-(tert-butoxycarbonyl)(4′-maleimidobutyl)amino)-4-methylpentyl)-thiazole-4-carboxylicacid (276)

To the dryness compound (275) (800 mg, 1.29 mmol) in a mixture of1,2-dichloroethane (40 ml) and toluene was added trimethyltin hydroxide(400 mg, 2.21 mmol). The mixture was stirred at 100° C. overnight,filtered through a short SiO₂ column, washed the column withMeOH/CH₂Cl₂/HOAc (1:5:0.01) and evaporated to dryness. To the crudedryness mixture in pyridine (15 ml) was added Ac₂O (0.3 ml, 3.17 mmol)at 0° C. The mixture was stirred at RT overnight, evaporated andpurified on SiO2 column eluted with MeOH/CH₂Cl₂/HOAc (1:10:0.01) toafford the title compound (578.4 mg, 74%). ESI: m/z: [M+Na]+, calcd forC₂₉H₃₉N3NaO₉S, 628.24, Found, 628.24.

Example 63. Phenylalanine-ketoepoxide

N-Boc phenylalanine-ketoepoxide (Sun, L. et al, J. Mol. Catalysis A:Chem., 2005, 234 (1-2), 29-34) (300 mg, 1.08 mmol) in 1,4-dioxane (8 mL)at 0° C. was added hydrochloric acid (37%, 2 mL). The mixture wasstirred for 1 hour at which time TLC showed complete consumption ofstarting material. The resulting solution was diluted with toluene (10ml), evaporated and crystallized with EtOH/Hexane to yield HCl salt ofthe title compound (201 mg, 87%). ESI: m/z: [M+H]+, calcd for C₁₁H₁₆NO,178.12, Found, 178.12.

Example 64. (S,E)-Ethyl5-phenyl-4-(tert-butoxy-carbonylamino)-2-methylpent-2-enoate (327)

L-t-Boc-phenylalanine methyl ester 326 (5.60 g, 20.05 mmol) in CH₂Cl₂(80 ml) at −78° C. was added dropwise DIBAL (40 ml, 40 mmol, 1.0 M) inCH₂Cl₂. After stirred at −78° C. for 45 min, the ylide solution preparedfrom 1-(1-ethoxycarbonyl ethyl)-triphenylphosphonium bromide (18.0 g,40.64 mmol) and KOtBu (5.00 g, 44.64 mmol) in CH₂Cl₂ (80 ml) at RT wasadded at −78° C. After stirred at −78° C. for 1 h and RT over night, themixture was poured into 1 L of NaH₂PO₄ (sat.) solution with vigorouslystirring. Separated and the aqueous phase was extracted with CH₂Cl₂. Theorganic layers were dried over Na₂SO₄, concentrated and purified by SiO₂chromatography (EtOAc/Hexane, 1:7-1:5) to afford 5.50 g (83% yields) ofthe title compound. ESI: m/z: [M+Nat]⁺, calcd for C₁₉H₂₇NNaO₄, 356.19,Found, 356.20.

Example 65. Ethyl3-((S)-1-(tert-butoxycarbonylamino)-2-phenylethyl)-2-methyloxirane-2-carboxylate(328)

The compound 327 (5.0 g, 15.0 mmol) in CH₂Cl₂ (80 ml) was added3-chloroperoxy-benzoic acid (5.5 g, 22.3 mmol) and the mixture wasstirred overnight, diluted with NaHCO₃ (25 ml, sat.), separated andextracted the aqueous solution with CH₂Cl₂. The organic layers werecombined, dried over Na₂SO₄, filtered, evaporated and purified on SiO2column (1:4 EtOAc/Hexane) to afford 4.71 g (90% yield) of the titlecompound. ESI: m/z: [M+Na]+, calcd for C₁₉H₂₇NNaO₅, 372.19, Found,372.20.

Example 66.3-((S)-1-(tert-butoxycarbonylamino)-2-phenylethyl)-2-methyloxirane-2-carboxylicacid (329)

To a solution of LiOH (5.0 g, 208.7 mmol) in H₂O (60 mL) was added to asolution of compound (328) (4.70 g, 13.45 mmol) in THF (100 mL). Afterstirred for 1 h, the mixture was concentrated, poured into H₂O (150 mL)and the mixture was adjusted to pH˜4 with 4M HCl aq. The mixture wasextracted with EtOAc, dried over Na₂SO₄, concentrated and purified bySiO₂ chromatography (MeOH/DCM/HOAc 1:10:0.01) to afford (3.97 g, 92%) ofthe title compound. ESI: m/z: [M+Na]⁺, calcd for C₁₇H₂₃NNaO₅, 344.16,Found 344.16.

Example 67.3-((S)-1-(((9H-fluoren-9-yl)methoxy)carbonylamino)-2-phenylethyl)-2-methyloxirane-2-carboxylicacid (331)

To a solution of compound (329) (3.90 g, 12.14 mmol) in CH₂Cl₂ (40 ml)at 0° C., was added TFA (10 ml) and the mixture was stirred at 0° C. for30 min, diluted with toluene, evaporated to dryness to form the crudeTFA salt of compound 330. In a solution of Na₂CO₃ (5.0 g, 47.16 mmol) inmixture of H₂O (60 mL) and ethanol (30 ml) were added the crude compound330 and Fmoc-Cl (3.70 g, 14.30 mmol). After stirred for 6 h, the mixturewas concentrated, poured into H₂O (150 mL) and the mixture was adjustedto pH˜4 with 4M HCl aq. The mixture was extracted with EtOAc, dried overNa₂SO₄, concentrated and purified by SiO₂ chromatography (MeOH/DCM/HOAc1:10:0.01) to afford 3.87 g (72% yields in 2 steps) of the titlecompound. ESI: m/z: [M+Na]⁺, calcd for C₂₇H₂₅NNaO₅, 466.17, Found466.17.

Example 68. General Solution Peptide Coupling Procedure

The HCl salt of an amine (1 eq) was dissolved in CH₂Cl₂ or DMF (0.2 M)and cooled to 4° C. in an ice bath, followed by the addition of theappropriate Boc-protected amino acid (1.3 eq), EDC (2 eq), or TBTU (2eq), or PyBrOP (2 eq), HOBt (1.5 eq) and DIPEA (3.5 eq). The reactionwas allowed to slowly warm to room temperature and stirred for 15 h,after which it was diluted with EtOAc and washed successively withaqueous solutions of 1M HCl, saturated sodium bicarbonate, water andsaturated sodium chloride. The organic layer was dried with Na2SO4,filtered and concentrated under reduced pressure. Purification by columnchromatography (0% to 20% MeOH:CH₂Cl₂) yielded Boc protected peptide.

Example 69. General Boc Deprotection Procedure

The Boc protected amino acid was dissolved in 20% TFA in CH₂Cl₂ or 4 MHCl in dioxane and stirred 30 min, or until the reaction was deemedcomplete by TLC. The solution was then concentrated under reducedpressure to give the TFA or HCl salt of the peptide. The TFA salt of thepeptide was coevaporated with 2% HCl in CH₂Cl₂/Toluene for 3-4 times togenerate the HCl salt.

Example 70. General Solid Phase Peptide Synthesis (SPPS) Procedure

Boc SPPS was used Merrifield resin or modified PAM resin or MBHA resin.Fmoc SPPS was used Wang resin, or 2-chlorotrityl resin, or HMPB, MBHAresin. The pre-treatment of the resin (pre-swell) and the first loadingof an amino compound were followed the manufacture's labels or notes.Resin bound Boc protected amino acids were deprotected with 20% TFA inCH₂Cl₂ or 3M HCl in dioxane for 30 minutes and washed with DMF, MeOH,50% DIPEA (iPr₂Net) in CH₂Cl₂ and CH₂Cl₂. For steps involving thedeprotection of multiple free amines, this step was repeated once beforeacylation to ensure completeness of reaction. Resin bound Fmoc protectedamino acids were deprotected with 20% piperidine in DMF for 30 minutesand washed with DMF, MeOH and CH₂Cl₂. For steps involving thedeprotection of multiple free amines, this step was repeated once beforeacylation to ensure completeness of reaction. The free amine beads werethen suspended in a solution of the protected amino acid (3 eq per eq offree amine), TBTU or PyBrOP (3 eq per eq of free amine) and DIPEA (5 eqper eq of free amine) and mixed for 4 h, and then washed with DMF, MeOHand CH₂Cl₂. For steps involving the acylation of multiple free amines,the coupling procedure was repeated once before deprotection to ensurecompleteness of reaction. These steps were repeated until the desiredpeptide was synthesized.

Example 71. General Cleavage of Peptides from Wang Resin or2-Chlorotrityl Resin

The peptide bound with Wang resin was mixed with 50% TFA in CH₂Cl₂ andtri-isopropylsilane (1-5%) or the peptide bound with 2-Chlorotritylresin was mixed with 1% TFA in CH₂Cl₂. Mixed for 2 h and then filtered.The resin was washed with CH₂Cl₂ (3×30 ml), Methanol (3×30 ml), whichwere combined with the filtrate and evaporated to almost dryness. ColdEt₂O was then added to precipitate the desired deprotected peptide.

Example 72. General Cleavage of Peptides from Merrifield, MBHA or PAMResin

The peptide bound with the resins was mixed with HF/Me₂S/anisole(10:1:1) or CH₃SO₃H/Me₂S/anisole (20:1:1), orHF/anisole/Me2S/p-thiocresol (10:1:1:0.2) for peptide containing Cys.Mixed for 2 h, concentrated under a stream of N2, diluted with TFA andfiltered. The resin was washed with CH₂Cl₂ (3×30 ml), Methanol (3×30ml), which were combined with the filtrate and condensed under reducedpressure to almost dryness. Cold Et₂O was then added to precipitate thedesired deprotected peptide.

Example 73. Chromatographic Purification

The crude peptide mixture was then purified through SiO₂ columnchromatography (10% to 25% MeOH in CH₂Cl₂) or by a reverse phase HPLCeluted with gradient from 100% of water (optionally containing 1% HOAc)to 30% of water (containing 1% HOAc)/70% methanol in 1 h, pooled thefraction, evaporated to give the desired protected.

Example 74. Conjugate Preparation

A binding molecule, preferably an antibody can be conjugated to anantimitotic agents of this prevention through amide, or thiol ether ordisulfide bond linkage. In a experiment of generating free thiols on anantibody, the antibodies (mAbs) (>5 mg/mL) in PBS containing 50 mMsodium borate, pH 8.0, were treated with dithiothreitol (10 mM final) at35° C. for 30 min. After gel filtration (G-25, PBS containing 1 mMEDTA), thiol determination using Ellman's reagent[5,5′-dithiobis(2-nitrobenzoic acid)] indicated that there wereapproximately eight thiols per mAb. The free thiols were also introducedthrough conjugation of Traut's Reagent (2-Iminothiolane) (Jue, R., etal. Biochem. 1978, 17 (25): 5399-5405), or through conjugation of SATP(N-succinimidyl-S-acetylthiopropionate) or SAT(PEG)4 linkers at pH 7-8,followed by releasing SH group with hydroxylamine treatment (Duncan, R,et al, Anal. Biochem. 1983, 132, 68-73, Fuji, N. et al, Chem. Pharm.Bull. 1985, 33, 362-367). On average, 5-8 of free thiols were introducedon mAbs.

To the mAbs containing free thiols at 4° C. were added the drug bearingmaleimide or bromoacetamide (0.5 M sodium borate buffer pH 9 wasrequired to promote mAb alkylation with bromoacetamide) (1.2˜1.5 equivof drug derivatives/SH group ratio) in cold DMA (2˜20% v/v). After 1˜2h, the reactions were quenched with excess cysteine; the conjugates wereconcentrated by centrifugal ultrafiltration, gel filtered (G-25, PBS),and sterile filtered. Protein concentration and drug loading weredetermined by spectral analysis at 280 and 252 nm, respectively.Size-exclusion HPLC was used to determine percent monomer of eachconjugate prepared, and RP-HPLC established that there was less than0.5% unconjugated cysteine-equenched drug. The resulting conjugate wasmonomeric and contained, on the average, 3.2-4.2 antimitotic agentslinked per antibody molecule for these thiol ether linked conjugation.

For the conjugation through DMPS, SMDP, SMPT, SPP, SPDP, SPDB, SMCC, orSM(PEG)n linkers. A solution of mAb (>5 mg/mL) in aqueous buffer (50 mMPBS, 50 mM NaCl, 1 mM EDTA) at pH 6.5˜7.5 was incubated for 2 h with a6- to 10-fold molar excess of a linker. The reaction mixture waspurified via a Sephadex G25 gel filtration column to remove lowmolecular weight material. (The concentration of the antibody wasdetermined spectro-photometrically if the linkers contained apyridylthio. The coefficients for the antibody ε_(280nm)=2067550 M⁻¹cm⁻¹. An aliquot of the modified antibody was treated with an excess(>20 equiv) of dithiothreitol and the release of pyridine-2-thionedetermined using the known extinction coefficients of ε_(343 nm)=8080M⁻¹ cm⁻¹ and ε_(280nm)=5100 M⁻¹ cm⁻¹ for pyridine-2-thione). Themodified antibody was treated with 1.2˜1.5 equiv of an antimitotic agentbearing a thiol group. The reaction mixture was incubated for 5˜18 h atRT. The reaction mixture was purified via a Sephadex G25 gel filtrationcolumn to remove the unconjugated drug and other low molecular weightspecies. The concentration of the conjugate was determinedspectrophotometrically at 280 and 252 nm. The resulting conjugate wasmonomeric and contained, on the average, 3.2-4.5 antimitotic agentmolecules linked per antibody molecule.

Example 75. In Vitro Cytotoxicity Assays

BJAB (Burkitt's lymphoma), BT-474 (breast carcinoma) cells, Namalwa(human Burkitt's lymphoma), Ramos (human Burkitt's lymphoma), COLO 205(human colon adenocarcinoma), and A375 (human malignant melanoma) werefrom ATCC. The breast tumor line KPL-4 was from Dr. J. Kurebayashi(Kurebayashi, J. et al. Br J Cancer 1999; 79: 707-17). The cultures weremaintained in RPMI 1640 supplemented with 10% heat-inactivated fetalbovine serum (FBS). All cell lines were cultured in a humidifiedincubator at 37° C., 6% CO₂. The cytotoxicity study was performed usinga clonogenic assay similar to a reference described (Franken, et al,Nature Protocols 1, 2315-2319 (2006)). The test cell lines were platedinto 6-well culture dishes at a constant number of 5000 cells per well.The cells were incubated with varying concentrations (1 pM to 50 nM) ofthe test-agent (the antimitotic agents or their conjugates) for 72 h.The medium was then aspirated from the plates and replaced with freshmedium. The cultures were allowed to grow and form colonies for a totalof 7 to 10 days after plating. The cultures were then fixed and stainedwith 0.2% crystal violet in 10% formalin/PBS, and the colonies werecounted. Plating efficiency of non-treated cells (medium alone) wasdetermined by dividing the number of colonies counted by the number ofcells plated. The surviving fraction of cells exposed to a toxic agentwas determined by dividing the number of colonies in wells that wereexposed to the agent by the number of colonies in the control wells.

REFERENCES

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1. A conjugate having a structure of Formula (F):

or a pharaceutically acceptable salt or solvate thereof, wherein T is acell-binding ligand, a dendrimer or a liposome: L is a releasablelinker; ----- is a linkage bond that L connects to an atom in a moietyinside the bracket independently; a is 1˜20 and m is 1˜10; wherein thecell-binding ligand is an antibody, or an antibody fragment that bindsto a target cell; wherein the releasable linker L has a formula of:-W_(w)-(Aa)_(r)-V_(v)- wherein: -W- is a stretcher unit, which links themoiety inside the bracket to an amino acid unit (Aa) when present, or Vwhen an Aa is not present, and W is independently a self-immolativespacer, a peptidyl unit, a hydrazone, a disulfide, a thioether, anester, or an amide bond; w is 0 or 1; wherein Aa is independently anatural or unnatural amino acid unit; r is independently an integerranging from 0 to 12; (Aa), represents a natural or unnatural aminoacid, dipeptide, tripeptide, letrapeptide, pentapeptide, hexapeptide,hptaeptaide, octapeptide, nonapeptide, decapeptide, undecapeptide ordodecapeptide unit; wherein V is a spacer unit and is independently H,O, NH, S, C₁-C₈ alkyl, C₂-C₈ heteroalkyl, alkenyl, or alkynyl, C₃-C₈aryl, heterocyclic, carbocyclic, cycloalkyl, alkylcycloalkyl,heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkyl, oralkyicarbonyl, or one to four amino acid units, or (CH₂CH₂O)_(r), r isan integer ranging from 0 to 12; and v is 0, 1 or 2; wherein R¹, R², R³,and R⁴ are independently H, C₁˜C₈ alkyl; C₂˜C₈ heteroalkyl orheterocyclic; C₃˜C₈ aryl, Ar-alkyl, cycloalkyl, alkylcycloalkyl,heterocycloalkyl, heteroalkylcycloalkyl, carbocyclic, or alkylcarbony;or R¹ and R², R¹ and R³, R² and R³, or R³ and R⁴ form a 3˜7 memberedcarbocyclic, cycloalkyl, heterocyclic, or heterocycloalkyl ring system;wherein Y is N or CH; wherein R⁵, R⁶, R⁸ and R¹⁰ are independently H, orC₁˜C₄ alkyl, or heteroalkyl: or R⁵ and R⁶ form a 3˜7 memberedcarbocyclic, cycloalkyl, heterocyclic, or heterocycloalkyl ring system;wherein R⁷ is independently H, R¹⁴, —R¹⁴C(═O)X¹R¹⁵, or —R¹⁴X¹R¹⁵,wherein X is O, S, S—S, NH, or NR¹⁴, and wherein R¹⁴ and R¹⁵ areindependently C₁˜C₈ alkyl, heteroalkyl; C₂˜C₈ alkenyl, alkynyl,heterocyclic; or C₃˜C₈ aryl, carbocyclic, cycloalkyl, heterocycloalkyl,heteroaralkyl, heteroalkylcycloalkyl, alkylcarbonyl; wherein R⁹ isindependently H, —OH, —OR¹⁴, —OC(═O)R¹⁴, —OC(═O)NHR¹⁴, —OP(═O)(OR¹⁴)₂,or —OR¹⁴OP(═O)(OR¹⁵)₂, wherein R¹⁴ and R¹⁵ are independently C₁˜C₈alkyl, heteroalkyl; C₂˜C₈ alkenyl, alkynyl, heterocyclic; or C₃˜C₈ aryl,carbocyclic, cycloalkyl, alkylcycloalkyl, heterocycloalkyl,heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl; wherein R¹¹ isindependently H, —R¹⁴, —R¹⁴C(═O)R¹⁶, —R¹⁴X²R¹⁶, —R¹⁴—, —R¹⁴C(═O)R¹⁷—,—R¹⁴X²R¹⁷—, or —R¹⁴C(═O)X²—, wherein X² is —O—, —S—, —NH—, —N(R¹⁴)—,—O—R¹⁴—, —S—R¹⁴—, —S(═O)—R¹⁴—, or —NHR¹⁴—, and wherein R¹⁴ is C₁˜C₈alkyl, heteroalkyl; C₂˜C₈ alkenyl, alkynyl, heterocyclic; or C₃˜C₈ aryl,carbocyclic, cycloalkyl, alkylcycloalkyl, heterocycloalkyl,heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl; R¹⁶ is H, OH, R¹⁴or one to four amino acid units; R¹⁷ is H, OH, C₁˜C₈ alkyl, orheteroalkyl; C₂˜C₈ alkenyl, alkynyl, or heterocyclic; C₃˜C₈ arylene,heterocyclic, carbocyclic, heterocycloalkyl; or an amino acid, or twoamino acid units; wherein R¹² is independently R¹⁴, —O—, —S—, —N—, ═N—,═NNH—, —OH, —SH, —NH₂, ═NH, ═NNH₂, —NH(R¹⁴), —OR¹⁴, —N(R¹⁴)—, —OR¹⁴—,—C(O)O—, —C(O)NHR¹⁴—, —SR¹⁴—, —S(═O)R¹⁴—, —NHR¹⁴—, —CH₂OP(═O)(OR¹⁵)—,—P(═O)(OR¹⁵)—, —C(O)OP(═O)(OR¹⁵)—, —OP(═O)(OR¹⁵)O—, or —SO₂R¹⁴—,—C(O)OR¹⁶—, —COR¹⁶, —COOR¹⁴—, —C(O)NH—, —C(O)NH₂, —C(O)NHR¹⁴, —SR¹⁴,—S(═O)R¹⁴, —P(═O)(OR¹⁶)₂, —OP(═O)(OR¹⁶)₂, —CH₂OP(═O)(OR¹⁶)₂, or —SO₂R¹⁶,wherein R¹⁴ and R¹⁵ are H, C₁˜C₈ alkyl, heteroalkyl; C₂˜C₈ alkenyl,alkynyl, heterocyclic; or C₃˜C₈ aryl, cycloalkyl, carbocyclic,alkylcycloalkyl, heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkyl,alkylcarbonyl; R¹⁶ is H, OH, R¹⁴ or one to four amino acid units;wherein R¹³ is C₁˜C₁₀ alkyl, heteroalkyl, alkyl acid, alkyl amide, alkylamine, or Ar; Ar is an aromatic or hetero aromatic group, composed ofone or several rings, and comprising four to ten carbon atoms; thehetero aromatic group is an aromatic group that has one or severalcarbon atoms replaced by hetero atoms; wherein in the aromatic group,one or several H atoms are optionally replaced independently by R¹⁷, F,Cl, Br, I, OR¹⁶, SR¹⁶, NR¹⁶R¹⁷, N═NR¹⁶, N═R¹⁶, NR¹⁶R¹⁷, NO₂, SOR¹⁶R¹⁷,SO₂R¹⁶, SO₃R¹⁶, OSO₃R¹⁶, PR¹⁶R₁₇, POR¹⁶R¹⁷, PO₂R¹⁶R¹⁷, OP(O)(OR¹⁷)₂,OCH₂OP(O)(OR¹⁷)₂, OC(O)OP(O)(OR¹⁷)₂, PO(OR¹⁶)(OR¹⁷),OP(O)(OR¹⁷)OP(O)(OR¹⁷)₂, OC(O)R¹⁷ or OC(O)NHR¹⁷, wherein R¹⁶ and R¹⁷ areindependently H, OH, C₁˜C₈ alkyl, or heteroalkyl; C₂˜C₈ alkenyl,alkynyl, or heterocyclic; C₃˜C₈ aryl, cycloalkyl, alkylcycloalkyl,carbocyclic, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl,alkylcarbonyl, or C₄˜C₁₂ glycoside, or a pharmaceutical salt; or R¹² andR¹³ form a 3˜7 membered carbocyclic, cycloalkyl, heterocyclic, aromaticor heterocycloalkyl ring system.
 2. The conjugate according to claim 1,having a structure of Formula (V):

or a pharmaceutically acceptable salt or solvate thereof, wherein T, L,n, m, Y, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ and R¹³ are defined thesame as in claim 1; wherein R¹¹ independently —R¹⁴—, —R¹⁴C(═O)R¹⁷,—R¹⁴X²R¹⁷—, or —R¹⁴C(═O)X²—, wherein R¹⁷ is independently C₁˜C₈ alkyl;C₂˜C₈ alkenyl, alkynyl, or heteroalkyl; C₃˜C₈ aryl, arylene,heterocyclic, carbocyclic, or heterocycloalkyl, or an amino acid, or twoamino acid; X² is —O—, —S—, —NH—, —N(R¹⁴)—, —O—R¹⁴—, —S—R¹⁴—,—S(═O)—R¹⁴—, or —NHR¹⁴—; R¹⁴ is C₁˜C₈ alkyl, or heteroalkyl; C₂˜C₈alkenyl, alkynyl, or heterocyclic; or C₃˜C₈ aryl, carbocyclic,cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl,heteroaralkyl, or alkylcarbonyl; wherein R¹² is independently R¹⁴, —OH,—SH, —NH, ═NH, ═NNH₂, —NH(R¹⁴), —OR¹⁴, —COR¹⁶, —C(O)NH₂, —C(O)NHR¹⁴,—SR¹⁴, —S(═O)R¹⁴, —P(═O)(OR¹⁶)₂, —OP(═O)(OR¹⁶)₂, —CH₂OP(═O)(OR¹⁶)₂, or—SO₂R¹⁶; wherein R¹⁴ is H, C₁˜C₈ alkyl, heteroalkyl; C₂˜C₈ alkenyl,alkynyl, heterocyclic; C₃˜C₈ aryl, cycloalkyl, carbocyclic,alkylcycloalkyl, heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkyl,alkylcarbonyl, and R¹⁶ is H, OH, R¹⁴ or one to four amino acid units. 3.The conjugate according to claim 1, having a structure of Formula (VI):

or a pharmaceutical acceptable salt or solvate thereof, wherein T, L, n,R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹³ are defined the same asin claim 1; wherein R¹¹ is independently H, —R¹⁴, —R¹⁴C(═O)R¹⁶,—R¹⁴X²R¹⁶, wherein X² is —O—, —S—, —NH—, —N(R¹⁴)—, —O—R¹⁴—, —S—R¹⁴,—S(═O)—R¹⁴—, or —NHR¹⁴—; R¹⁴ is C₁˜C₈ alkyl, heteroalkyl, C₂˜C₈ alkenyl,alkynyl, heterocyclic; C₃˜C₈ aryl, cycloalkyl, carbocyclic,alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl,alkylcarbonyl; R¹⁶ is H, OH, R¹⁴ or one to four amino acid units;wherein R¹² is independently —R¹⁴, —O—, —S—, —NH—, ═N—, ═NNH—, —N(R¹⁴)—,—OR¹⁴—, —C(O)O—, —C(O)NH—, —C(O)NHR¹⁴—, —SR¹⁴—, —S(═O)R¹⁴—, —NHR¹⁴—,—CH₂OP(═O)(OR¹⁵)—, —P(═O)(OR¹⁵)—, —C(O)OP(═O)(OR¹⁵)—, —OP(═O)(OR¹⁵)O—,or —SO₂R¹⁴—; R¹⁴ and R¹⁵ are independently H, C₁˜C₈ alkyl, orheteroalkyl; C₂˜C8 alkenyl, alkynyl, or heterocyclic; or C₃˜C₈ aryl,carbocyclic, cycloalkyl, alkylcycloalkyl, heterocycloalkyl,heteroaralkyl, heteroalkylcycloalkyl, or alkylcarbonyl; provided thatR¹² is not H.
 4. The conjugate according to claim 1, having a structureof Formula (VII):

or a pharmaceutically acceptable salt solvate thereof, wherein T, L, n,m, Y, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are defined the sameas in claim 1; wherein R¹¹ is independently H, —R¹⁴, —R¹⁴C(═O)R¹⁶,—R¹⁴X²R¹⁶, wherein X² is —O—, —S—, —NH—, —N(R¹⁴)—, —O—R¹⁴—, —S—R¹⁴—,—S(═O)—R¹⁴—, or —NHR¹⁴—; R¹⁴ is C₁˜C₈ alkyl, heteroalkyl; C₂˜C₈ alkenyl,alkynyl, heterocyclic; or C₃˜C₈ aryl, cycloalkyl, carbocyclic,alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl,alkylcarbonyl; R¹⁶ is H, OH, R¹⁴ or one to four amino acid units;wherein R¹² is independently R¹⁴, —OH, —SH, —NH₂, ═NH—, ═NNH₂,—NH(R¹⁴)—, —OR¹⁴—, —COR¹⁶, C(O)NH₂, C(O)NHR¹⁴, —SR¹⁴, —S(═O)R¹⁴,—P(═O)(OR¹⁶)₂, —OP(═O)(OR¹⁶)₂, —CH₂OP(═O)(OR¹⁶)₂, or —SO₂R¹⁶, whereinR¹⁴ is H, C₁˜C₈ alkyl, or heteroalkyl; C₂˜C8 alkenyl, alkynyl,heterocyclic; or C₃˜C₈ aryl, cycloalkyl, carbocyclic, alkylcycloalkyl,heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkyl, alkylcarbonyl;R¹⁶ is H, OH, R¹⁴ or one to four amino acid units; wherein R¹³ is C₁˜C₁₀alkyl, heteroalkyl, alkyl acid alkyl amide, alkyl amine, or Ar; Ar is anaromatic or hetero aromatic group, composed of one or several rings, andcomprising four to ten carbon atoms; the hetero aromatic group is anaromatic group that has one or several carbon atoms replaced by heteroatoms; Ar is an aromatic group, wherein one or several H atoms areoptionally replaced independently by R¹⁸, F, Cl, Br, I, OR¹⁶, SR¹⁶,NR¹⁶R¹⁷, N═NR¹⁶, N═R¹⁶, R¹⁶R¹⁸, NO₂, SOR¹⁶R¹⁸, SO₂R¹⁶, SO₃R¹⁶, OSO₃R¹⁶,PR¹⁶R¹⁸, POR¹⁶R¹⁸, PO₂R¹⁶R¹⁸, OPO₃R¹⁶R¹⁸, CH₂OPO₃R¹⁶R¹⁸,OP(O)(OR¹⁶)OP(O)(OR¹⁸)₂, C(O)OPO₃R¹⁶R¹⁸, or PO₃R¹⁶R¹⁸, wherein R¹⁶, R¹⁸are independently H, C₁˜C₈ of alkyl; C₂˜C₈ of alkenyl, alkynyl, orheteroalkyl; C₃˜C₈ aryl, heterocyclic, carbocyclic, cycloalkyl,alkylcycloalkyl, heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkyl,or alkylcarbonyl; or C₄˜C₁₂ glycoside; or a pharmaceutical salt.
 5. Theconjugate according to claim 1, having a structure of formula Va, Vb,Vc, Vd, Ve, Vf, Vg, Vh, VIa, VIb, VIc, VIe, VIf, VIg, VIh, VIi, VIIa,VIIb, VIIc, VIId, VIIe, VIIf, VIIg, VIIh, VIIi, VIIj, VIIk, VIIl, VIIm,VIIn, VIIo, VIIq, VIIr, VIIs, or VIIt, which are illustrated below:

wherein Aa is a natural or unnatural amino acid; Ar is aryl; n is 1˜20;q=1˜5; X′, Y′ and Z′ are independently CH, O, S, NH, or NR²²; R²², R²³and R²⁴ are independently C₁˜C₈ alkyl; C₂˜C₈ alkenyl, alkynyl, orheteroalkyl; C₃˜C₈ aryl, heterocyclic, carbocyclic, cycloalkyl,alkylcycloalkyl, heterocycloalkyl, Ar-alkyl, heteroalkylcycloalkyl, orheteroaralkyl, or —(OCH₂CH₂)_(n); R′ and R″ are independently H or CH₃.6. The conjugate according to claim 1, having a structure of formula:mAb-TZ01, mAb-TZ04, mAb-TZ05, mAb-TZ07, mAb-TZ10a, mAb-TZ10b, mAb-TZ11,mAb-TZ12, mAb-TZ13, mAb-TZ14, mAb-TZ17, mAb-TZ19, mAb-TZ20, mAb-TZ21,mAb-TZ22, mAb-TZ23, mAb-TZ24, or mAb-TZ25, which are illustrated below:

wherein mAb is an antibody.
 7. The conjugate of claim 1, wherein thecell-binding ligand is capable of binding to target cells selected fromthe group consisting of tumor cells, autoimmune cells, myeloid cells, Bcells, and melanocytes.
 8. The conjugate according to claim 1, whereinwhen n in Formula (I′) is 2, the linker L has following formula:

wherein R is C₁˜C₈ alkyl or C₂H₄(OC₂H₄)_(n), n is 1˜20.
 9. The conjugateof claim 1, wherein when n in Formula (I′) is 2, the conjugate hasfollowing formula:

wherein R is C₁˜C₈ alkyl or C₂H₄(OC₂H₄)_(n), n is 1˜20; D is a cytotoxicagent, which is a moiety inside the bracket of Formula (I), T is acell-binding ligand.
 10. The conjugate of claim 1, wherein when n inFormula (I′) is 2, the conjugate has following formula:

wherein R is C₁˜C₈ alkyl or C₂H₄(OC₂H₄)_(n), n is 1˜20; D is a cytotoxicagent, which is a moiety inside the bracket of Formula (I′), T is acell-binding ligand.
 11. The conjugate of claim 10, wherein at least oneof Ds is selected from the group consisting of calicheamicins,auristatins, maytansinoids, dolastatins, CC-1065 compounds,doxorubicins, taxanes, pyrrolobenzodiazepine dimer, siRNA and acombination thereof, or a pharmaceutically acceptable salt, acid orderivatives thereof.
 12. A pharmaceutical composition comprising atherapeutically effective amount of the conjugate of claim 1, or apharmaceutically acceptable salt thereof, and a carrier, diluent, orexcipient therefor.
 13. A method for preparing the conjugate accordingto claim 1, comprising reacting a cell binding agent with a moleculehaving a structure of formula: 232a, 232b, 232c, 232d, 232e, 232f, 232g,232h, 232i, 232j, 232k, 232l, 232m, 232n, 232o, 232p, 232q, 232r, 232s,232t, 221a, 221b, 233a, 233b, 233c, 233d, 222a, 241, 242, 247, 248, 206,211, 346, 350, 354, 357, 361, 366, 371, or 376, which are illustratedbelow:


14. The conjugate of claim 1, wherein the cell binding ligand is amonoclonal antibody, a single chain monoclonal antibody, a monoclonalantibody fragment that binds to the target cell, a chimeric antibody, ora chimeric antibody fragment that binds to the target cell.
 15. Theconjugate of claim 1, wherein the cell-binding ligand is capable ofbinding Apo2, BAFF-R, bone morphogenetic protein receptor, IGF-IR,CA125, CanAg, E16, ErbB2, MUC1, MUC16, Napi3b, TF, EpCAM, FcRH2, C242,CD2, CD3, CD4, CD5, CD6, CD11, CD18, CD19, CD20, CD21, CD22, CD26, CD30,CD33, CD37, CD38, CD40, CD44, CD56, CD70, CD72, CD79, CD90, CD105,CD138, CR, CR1, CRGF, CRIPTO, CXCR5, LY64, TDGF1, endothelin B receptor,EphA receptors, EphB receptors, Endothelin, FCRH1, HER2/neu, HER3, MHCclass II molecule Ia antigen, integrins, IRTA2, LIV-1, MPF, Napi2b,PDL1, FLJ10372, KIAA1445 Mm42015, SEMA5B, SEMAG, six transmembraneepithelial antigen of prostate 1, IPCA1, PCANP1, STMP, prostateantigens; insulin growth factor receptor, or folate receptor on targetcells.
 16. A pharmaceutical composition comprising the conjugate ofclaim 1, and a therapeutic agent selected from the group consisting ofchemotherapeutic agents, immunotherapy agents, and anti-autoimmunedisorder agents.
 17. A conjugate having a structure of Formula (VIII):

or a pharmaceutically acceptable salt or solvate thereof, wherein T is acell-binding ligand, a dendrimer or a liposome; L is a releasablelinker; ----- is a linkage bond that 1, connects to an atom in a moietyinside the bracket independently; n is 1˜20 and in is 1˜10; wherein thecell-binding ligand is an antibody, or an antibody fragment that bindsto a target cell; wherein the releasable linker L has a formula of:-W_(w)-(Aa)_(r)-V_(v)-, wherein: -W- is a stretcher unit, which linksthe moiety inside the bracket to an amino acid unit (Aa) when present,or V when an Aa is not present, and W is independently a self-immolativespacer, a peptidyl unit, a hydrazone, a disulfide, a thioether, anester, or an amide bond; w is 0 or 1; wherein Aa is independently anatural or unnatural amino acid unit; r is independently an integerranging from 0 to 12; (Aa)_(r) represents a natural or unnatural aminoacid, dipeptide, tripeptide tetrapeptide, pentapeptide, hexapeptide,heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide ordodecapeptide unit; wherein V is a spacer unit and is independently H,O, NH, S, C₁˜C₈ alkyl, C₂˜C₈ heteroalkyl, alkenyl, or alkynyl, C₃˜C₈aryl, heterocyclic, carbocyclic, cycloalkyl, alkylcycloalkyl,heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkyl, oralkylcarbonyl, or one to four amino acid units, or (CH₂CH₂O)_(r), r isan integer ranging from 0 to 12; and v is 0, 1 or 2; wherein q=1˜5;wherein Z¹ is H, CH₂OP(O)(OR¹⁸)₂, PO(OR¹⁸)₂, C(O)OP(O)(OR¹⁸)₂,P(O)(OR¹⁸)OP(O)(OR¹⁸)₂, C(O)R¹⁸, C(O)NHR¹⁸, SO₂(OR¹⁸), C₄˜C₁₂ glycoside,or C₁˜C₈ alkyl, carboxyalkyl, or heterocyclic; R¹⁸ is H, C₁˜C₈ alkyl, orheteroalkyl; C₂˜C₅ alkenyl, alkynyl, carboxyalkyl, or heterocyclic;C₃˜C₈ aryl, or alkylcarbonyl; and wherein R²⁴ and R^(24′) areindependently H or CH₃.
 18. The conjugate according to claim 2, wherein:(i) R¹² is H when R¹⁰ is not H, or (ii) R¹² is H when R¹³ is:

wherein Z¹ is H, CH₂OP(O)(OR¹⁸)₂, C(O)OP(O)(OR¹⁸)₂, PO(OR¹⁸)₂,P(O)(OR¹⁸)OP(O)(OR¹⁸)₂, C(O)R¹⁸, C(O)NHR¹⁸, SO₂(OR¹⁸), C₄˜C₁₂ glycoside,C₁˜C₈ alkyl, or C₃˜C₈ carboxyalkyl, or heterocyclic; R¹⁹ is H, OH, NH₂,OSO₂(OR¹⁸), XCH₂OP(O)(OR¹⁸)₂, XPO(OR¹⁸)₂, XC(O)OP(O)(OR¹⁸)₂, XC(O)R¹⁸,XC(O)NHR¹⁸, C₁˜C₈ alkyl, carboxylic acid derivative; C₂˜C₈ alkenyl,alkynyl, heterocyclic, or carboxyalkyl; C₃˜C₈ aryl, or alkylcarbonyl; ora pharmaceutical salt; X is O, S, or NH; Y¹ and Y² are N or CHindependently; and R¹⁸ is H, C₁˜C₈ alkyl, or heteroalkyl; C₂˜C₈carboxyalkyl, alkenyl, alkynyl, or heterocyclic; or C₃˜C₈ aryl, oralkylcarbonyl.
 19. The conjugate according to claim 4, wherein: (i) R¹²is H when R¹⁰ is not H, or (ii) R¹² is H when R¹¹ is:

wherein X² is O, S, or N—R⁸; R⁸ is H, C₁˜C₆ alkyl or heteroalkyl.
 20. Amethod for treatment of a cancer, an autoimmune disease, or aninfectious disease comprising administering to a patient in need thereofa pharmaceutical composition comprising a therapeutically effectiveamount of the conjugate according to claim
 1. 21. The method accordingto claim 20, further comprising administering concurrently asynergistically effective amount of a therapeutic agent selected fromthe group consisting of chemotherapeutic agents, radiation therapyagents, immunotherapy agents, anti-autoimmune disorder agents, andanti-infectious agents.